ArticleLiterature Review

Subunits of Voltage-Gated Calcium Channels

January 2004
Journal of Bioenergetics and Biomembranes 35(6):599-620

January 2004
35(6):599-620

DOI:10.1023/B:JOBB.0000008026.37790.5a

Source
PubMed

Authors:

Annette C Dolphin

University College London

Calcium channel beta subunits have marked effects on the trafficking and on several of the biophysical properties of all high voltage activated calcium channels. In this article I shall review information on the different genes, on the structure of the beta subunits, and on their differential expression and post-translational modification. Their role in trafficking and assembly of the calcium channel heteromultimer will be described, and I will then review their effects on voltage-dependent and kinetic properties, stressing the differences between palmitoylated beta2a and the other beta subunits. Evidence for effects on calcium channel pharmacology will also be examined. I shall discuss the hypothesis that beta subunits can bind reversibly to calcium channels, and examine their role in the G protein modulation of calcium channels. Finally, I shall describe the consequences of knock-out of different beta subunit genes, and describe evidence for the involvement of beta subunits in disease.

Behavioural and neuronal correlates of central pain processing in a rat model of osteoarthritis

Conference Paper

Oct 2019

Carlota Montagut‐Bordas

Osteoarthritis (OA) of the knee join is a chronic condition characterized by the loss of articular cartilage around the joint leading to changes in joint capsule. Clinically, OA is manifested by joint stiffness, swelling, bone tenderness, discomfort upon movement and joint pain. The latter is the main reason why patients seek medical treatment. OA pain is often classified as nociceptive due to tissue damage leading to inflammation. However, a subgroup of OA patients exhibits pain with neuropathic pain-like features. Thus, it is important to understand the differences in pain processing between these two groups of patients, as it will have implications on treatment. 2mg of monosodium iodoacetate (MIA) was intra-articularly injected into the left knee of male Sprague-Dawley rats in order to study behavioural and electrophysiological changes that appear during the development of OA pain. The MIA model provides two distinct stages of OA pain. An early acute inflammatory stage (2-4 days after MIA injection) and a late stage that presents neuropathic pain-like features (14-21 days post MIA injection). Cartilage damage scores differ between the early and late stage MIA animals with the latter exhibiting a more severe maximal OA score. Behaviourally, paw withdrawal thresholds decrease in the acute inflammatory stage and returned almost back to baseline in the late stage. Weight bearing deficits are present in both stages suggesting that both groups exhibit on going pain. CaV2.2 is present in the pre-synaptic terminals of primary afferent fibers in the spinal dorsal horn and mediates neurotransmitter release. ω-conotoxin GVIA is a small peptide that acts as a state independent blocker of CaV2.2, while TROX-1 is a state dependent blocker of the channel. In-vivo electrophysiological recordings of WDR neurones revealed that ω–conotoxin was able to significantly inhibit neuronal evoked responses to electrical, dynamic brush, mechanical and thermal stimuli in the late stage MIA animals but had little or no effects in the early stage MIA animals. State dependent blocker TROX-1, had no effects in neuronal responses of WDR neurones, in any group. Additionally, in-vivo electrophysiological revealed an increased descending serotonergic drive in a subgroup of late stage MIA animals. Additionally, Descending noxious inhibitory controls (DNIC) disappeared in this stage of the model as observed after neuropathy. Suggesting that in the late stage descending modulation is altered.

Molecular Sciences Presynaptic Calcium Channels

Article

May 2019

Sumiko Mochida

Presynaptic Calcium Channels

Article

Full-text available

May 2019
INT J MOL SCI

Sumiko Mochida

Presynaptic Ca2+ entry occurs through voltage-gated Ca2+ (CaV) channels which are activated by membrane depolarization. Depolarization accompanies neuronal firing and elevation of Ca2+ triggers neurotransmitter release from synaptic vesicles. For synchronization of efficient neurotransmitter release, synaptic vesicles are targeted by presynaptic Ca2+ channels forming a large signaling complex in the active zone. The presynaptic CaV2 channel gene family (comprising CaV2.1, CaV2.2, and CaV2.3 isoforms) encode the pore-forming α1 subunit. The cytoplasmic regions are responsible for channel modulation by interacting with regulatory proteins. This article overviews modulation of the activity of CaV2.1 and CaV2.2 channels in the control of synaptic strength and presynaptic plasticity.

Aberrant DJ-1 expression underlies L-type calcium channel hypoactivity in dendrites in tuberous sclerosis complex and Alzheimer’s disease

Article

Full-text available

Oct 2023

L-type voltage-gated calcium (Ca ²⁺ ) channels (L-VGCC) dysfunction is implicated in several neurological and psychiatric diseases. While a popular therapeutic target, it is unknown whether molecular mechanisms leading to disrupted L-VGCC across neurodegenerative disorders are conserved. Importantly, L-VGCC integrate synaptic signals to facilitate a plethora of cellular mechanisms; however, mechanisms that regulate L-VGCC channel density and subcellular compartmentalization are understudied. Herein, we report that in disease models with overactive mammalian target of rapamycin complex 1 (mTORC1) signaling (or mTORopathies), deficits in dendritic L-VGCC activity are associated with increased expression of the RNA-binding protein (RBP) Parkinsonism-associated deglycase (DJ-1). DJ-1 binds the mRNA coding for the alpha and auxiliary Ca ²⁺ channel subunits Ca V 1.2 and α2δ2, and represses their mRNA translation, only in the disease states, specifically preclinical models of tuberous sclerosis complex (TSC) and Alzheimer’s disease (AD). In agreement, DJ-1-mediated repression of Ca V 1.2/α2δ2 protein synthesis in dendrites is exaggerated in mouse models of AD and TSC, resulting in deficits in dendritic L-VGCC calcium activity. Finding of DJ-1-regulated L-VGCC activity in dendrites in TSC and AD provides a unique signaling pathway that can be targeted in clinical mTORopathies.

Presynaptic voltage-gated calcium channels in the auditory brainstem

Article

Mar 2021
MOL CELL NEUROSCI

Sound information encoding within the initial synapses in the auditory brainstem requires reliable and precise synaptic transmission in response to rapid and large fluctuations in action potential (AP) firing rates. The magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (CaV) in the presynaptic terminal are key determinants in triggering AP-mediated release. In the mammalian central nervous system (CNS), the CaV2.1 subtype is the critical subtype for CNS function, since it is the most efficient CaV2 subtype in triggering AP-mediated synaptic vesicle (SV) release. Auditory brainstem synapses utilize CaV2.1 to sustain fast and repetitive SV release to encode sound information. Therefore, understanding the presynaptic mechanisms that control CaV2.1 localization, organization and biophysical properties are integral to understanding auditory processing. Here, we review our current knowledge about the control of presynaptic CaV2 abundance and organization in the auditory brainstem and impact on the regulation of auditory processing.

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

Article

Full-text available

Feb 2008
PLOS ONE

The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity.

Identification and characterisation of rare CACNG5 genetic variants in bipolar disorder and schizophrenia

Thesis

Jun 2015

Y-C Lin

Schizophrenia (SCZ) and bipolar disorder (BPD) are common, highly heritability psychiatric disorders. Genome-wide association studies have found evidence of shared genetic susceptibility to both diseases. The most notable example is CACNA1C which encodes the 1 subunit of L-type calcium channels. Several other calcium channel genes have also been implicated in BPD and/or SCZ and together there is support for a role for these genes in both diseases. The primary function of several  subunit calcium channel genes appears to be the regulation of AMPA receptor localisation and function. Collectively these are known as Transmembrane AMPA receptor Regulatory Proteins (TARPs). This thesis aimed to identify disease relevant genetic variation in one such TARP, CACNG5, and to study the effect of these variants. CACNG5 variants in the exons and promoter region were identified in 1098 BPD, 618 SCZ, and 1087 control individuals. Four novel non-synonymous SNPs (nsSNPs) and four nsSNPs were identified. Burden analysis of nsSNPs in BPD and SCZ found evidence for association (p=0.0022). This association was strengthened by inclusion of data from European samples in the 1000 Genomes project (p=0.00057). However, combined data with the UK10K and Swedish exome sequence studies founds a weakened association signal (p=0.0082). Functional analyses using co-expression of AMPAR2 and CACNG5 constructs containing the eight nsSNPs were used to analyse changes in the expression and/or trafficking of 5 and AMPA receptors. Four of the variants were associated with decreased AMPAR2 expression as a consequence of altered trafficking to the cell surface. V146M (identified in 2 SCZ patients) overexpression increased AMPAR2 trafficking to the cell surface (p<0.005); conversely, T164L (identified in one SCZ patient) overexpression decreased the expression of AMPAR2 and its cell surface trafficking (p<0.05). Our results suggest a role for CACNG5 variants in SCZ and/or BPD and that this may be mediated via dysregulation of AMPARs

Physiology and Molecular Biology of Ion Channels Underlying Ventricular Repolarization of the Mammalian Heart

Chapter

Jan 2020

The physiology of the heart is controlled by an indigenous electrical system that regulates heart rhythm and contractile activity. The timing of cardiac electrical events is critical to proper heart function, and key to this activity is duration of the excitation of tissues in ventricular chambers. The duration of these events must be critically controlled: depolarization that is too brief presents vulnerability of the heart to premature excitation and a congenital disorder – the short QT syndrome. Prolonged depolarization underlies both congenital and drug-induced long QT syndrome that can lead to arrhythmias and sudden death. The critical electrical event in determining the duration of ventricular depolarization is the timing of repolarization of ventricular muscle cells. The timing is due to a critical balance of ion channel and electrogenic exchange currents such that a balance that tips in favor of the outward flow of positive ions begins the repolarization process. The molecular determinants of these currents are ions passing through a network of ion channels. In this chapter we review the key ion channels that underlie cardiac repolarization and focus on critical potassium ion channels. Multiple potassium channels contribute to this electrical activity, and together they underlie repolarization reserve of the heart. When this reserve is compromised by drug- or mutation-induced decreases in individual ion channels, risk of arrhythmia is increased. Here we focus on cardiac ionic currents, their molecular determinants, and the roles they play in cardiac repolarization.

Cardiac CaV1.2 channels require β subunits for β-adrenergic–mediated modulation but not trafficking

Article

Full-text available

Nov 2018
J CLIN INVEST

Ca2+ channel β-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant cardiac α1C subunits lacking capacity to bind CaVβ because of alanine-substitutions of three conserved residues - Y467, W470, and I471 in the α-interaction domain of rabbit α1C - can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these β-less Ca2+ channels cannot be stimulated by β-adrenergic pathway agonists, and thus adrenergic-augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a β-subunit-sequestering-peptide sharply curtailed β-adrenergic stimulation of wild-type Ca2+ channels, identifying an approach to specifically modulate β-adrenergic regulation of cardiac contractility. Our data demonstrate that β subunits are required for β-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.

Targeted Therapy for Orofacial Pain: A Novel Perspective for Precision Medicine

Article

Full-text available

Mar 2023

Orofacial pain (OFP) is a dental specialty that includes the diagnosis, management and treatment of disorders of the jaw, mouth, face, head and neck. Evidence-based understanding is critical in effectively treating OFPs as the pathophysiology of these conditions is multifactorial. Since OFP impacts the quality of life of the affected individuals, treating patients successfully is of the utmost significance. Despite the therapeutic choices available, treating OFP is still quite challenging, owing to inter-patient variations. The emerging trends in precision medicine could probably lead us to a paradigm shift in effectively managing the untreatable long-standing pain conditions. Precision medicine is designed based on the patient’s genetic profile to meet their needs. Several significant relationships have been discovered based on the genetics and genomics of pain in the past, and some of the notable targets are discussed in this review. The scope of this review is to discuss preclinical and clinical trials that include approaches used in targeted therapy for orofacial pain. Future developments in pain medicine should benefit from current trends in research into novel therapeutic approaches.

β subunits of voltage-gated calcium channels in cardiovascular diseases

Article

Full-text available

Feb 2023

Calcium signaling is required in bodily functions essential for survival, such as muscle contractions and neuronal communications. Of note, the voltage-gated calcium channels (VGCCs) expressed on muscle and neuronal cells, as well as some endocrine cells, are transmembrane protein complexes that allow for the selective entry of calcium ions into the cells. The α1 subunit constitutes the main pore-forming subunit that opens in response to membrane depolarization, and its biophysical functions are regulated by various auxiliary subunits–β, α2δ, and γ subunits. Within the cardiovascular system, the γ-subunit is not expressed and is therefore not discussed in this review. Because the α1 subunit is the pore-forming subunit, it is a prominent druggable target and the focus of many studies investigating potential therapeutic interventions for cardiovascular diseases. While this may be true, it should be noted that the direct inhibition of the α1 subunit may result in limited long-term cardiovascular benefits coupled with undesirable side effects, and that its expression and biophysical properties may depend largely on its auxiliary subunits. Indeed, the α2δ subunit has been reported to be essential for the membrane trafficking and expression of the α1 subunit. Furthermore, the β subunit not only prevents proteasomal degradation of the α1 subunit, but also directly modulates the biophysical properties of the α1 subunit, such as its voltage-dependent activities and open probabilities. More importantly, various isoforms of the β subunit have been found to differentially modulate the α1 subunit, and post-translational modifications of the β subunits further add to this complexity. These data suggest the possibility of the β subunit as a therapeutic target in cardiovascular diseases. However, emerging studies have reported the presence of cardiomyocyte membrane α1 subunit trafficking and expression in a β subunit-independent manner, which would undermine the efficacy of β subunit-targeting drugs. Nevertheless, a better understanding of the auxiliary β subunit would provide a more holistic approach when targeting the calcium channel complexes in treating cardiovascular diseases. Therefore, this review focuses on the post-translational modifications of the β subunit, as well as its role as an auxiliary subunit in modulating the calcium channel complexes.

CaV2.2 Channels in Brain Development and Synaptic Plasticity

Thesis

Sep 2022

Kjara S. Pilch

In the mammalian brain, presynaptic CaV2.2 channels play a pivotal role for synaptic transmission by mediating fast neurotransmitter exocytosis via influx of Ca2+ into the active zone at the presynaptic terminal. The distribution and activity of CaV2.2 channels at different synapses and maturity stages in the brain remains to be elucidated. In this study, I first show high levels of CaV2.2 channels in mouse cortex and hippocampus throughout development, persisting into adulthood. In contrast, CaV2.2 channels in the cerebellum and brain stem decreased as the brain matured. I thereafter assessed CaV2.2 channels during homeostatic synaptic plasticity, a compensatory form of homeostatic control preventing excessive or insufficient neuronal activity during which extensive active zone remodelling has been described. In this work I show that chronic silencing of neuronal activity in mature hippocampal cultures resulted in elevated presynaptic Ca2+ transients, mediated by a 30 % increase in CaV2.2 channel levels at the presynapse. Next, this work focussed on α2δ-1 subunits, important regulators of synaptic transmission and CaV2.2 channel abundance at the presynaptic membrane. Here, I show that α2δ-1- overexpression reduces the contribution of CaV2.2 channels to total Ca2+ flux without altering the amplitude of the Ca2+ transients. Finally, levels of endogenous α2δ-1 decreased during homeostatic synaptic plasticity, whereas the overexpression of α2δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV2.2 channels and novel roles for α2δ-1 during plastic synaptic adaptation.

Bipolar disorder associated miR-499-5p controls dendritic development and Cav1.2 calcium channel activity

Article

Jan 2021

Helena Sofia Caria Martins

Affective disorders, such as Major depressive disorder (MDD) and Bipolar disorder (BD), are a group of neuropsychiatric disorders characterized by rofound mood dysregulations. They constitute leading causes of disability and mortality, with especially high rates of suicide. However, the lack of understanding of the molecular mechanisms that trigger the development of affective disorders imposes a challenge to the discovery of novel and effective pharmacotherapies. It is currently accepted that a complex interaction between genetic predisposition and exposure to environmental stressors, such as childhood maltreatment, is required for the development of these disorders. The underlying causes, however, are only poorly understood. One of the main research areas focuses on the dysregulation of synaptic and neural plasticity, based on reports of altered structural and functional brain connectivity in the prefrontal cortex (PFC) and hippocampus of patients. Genes that are involved in neuronal development and plasticity are regulated by microRNAs (miRNAs or miRs), a class of small non-coding RNAs. Importantly, dysregulated miRNA expression is frequently observed in the brain and blood of MDD and BD patients, providing a rationale to consider miRNAs as therapeutic tools and disease biomarkers. This study aimed to investigate how changes in gene expression mediated by miRNAs interact with negative life events, especially during early life, and result in aberrant neuroplastic alterations that increase the susceptibility to affective disorders. For this purpose, I characterized the change in miR-499-5p expression in the blood of affective disorder patients and healthy individuals at higher risk of developing an affective disorder due to a history of childhood maltreatment. I found a significant up-regulation in circulating miR-499-5p in patients and maltreated subjects. Consistently, I observed higher levels of miR-499-5p in the hippocampus of a rat model of early life adversity. To understand how the dysregulation of miR499-5p causes neuronal abnormalities, I used primary cultures from the rat hippocampus to overexpress miR-499-5p and evaluate changes in neuronal morphology and function. In rat hippocampal neurons, miR-499-5p targets the Cacnb2 gene, the auxiliary β-subunit of the L-type Cav1.2 calcium channels, and a risk gene for psychiatric disorders. Elevated miR-499-5p expression inhibited Cacnb2 mRNA translation, impaired dendritic development, and reduced Cav1.2 surface expression and activity. Importantly, overexpression of miR-499-5p in the hippocampus induced short-term memory impairments in the Cacna1c+/- rat model. Based on these results, I propose a mechanism of miRNA-mediated calcium dysfunction in BD susceptibility whereby early life stress induces the expression of miR-499-5p, which in turn impairs dendritic development by inhibiting the expression of an auxiliary subunit of Cav1.2 calcium channels. My work further suggests that the increased blood expression of miR-499-5p could potentially be used as a biomarker of BD development and disease progression.

Elucidating the role of the L-type calcium channel in excitability and energetics in the heart: The ISHR 2020 Research Achievement Award Lecture

Article

Full-text available

Nov 2022
J MOL CELL CARDIOL

Livia C. Hool

Cardiovascular disease continues to be the leading health burden worldwide and with the rising rates in obesity and type II diabetes and ongoing effects of long COVID, it is anticipated that the burden of cardiovascular morbidity and mortality will increase. Calcium is essential to cardiac excitation and contraction. The main route for Ca²⁺ influx is the L-type Ca²⁺ channel (Cav1.2) and embryos that are homozygous null for the Cav1.2 gene are lethal at day 14 postcoitum. Acute changes in Ca²⁺ influx through the channel contribute to arrhythmia and sudden death, and chronic increases in intracellular Ca²⁺ contribute to pathological hypertrophy and heart failure. We use a multidisciplinary approach to study the regulation of the channel from the molecular level through to in vivo CRISPR mutant animal models. Here we describe some examples of our work from over 2 decades studying the role of the channel under physiological and pathological conditions. Our single channel analysis of purified human Cav1.2 protein in proteoliposomes has contributed to understanding direct molecular regulation of the channel including identifying the critical serine involved in the “fight or flight” response. Using the same approach we identified the cysteine responsible for altered function during oxidative stress. Chronic activation of the L-type Ca²⁺ channel during oxidative stress occurs as a result of persistent glutathionylation of the channel that contributes to the development of hypertrophy. We describe for the first time that activation of the channel alters mitochondrial function (and energetics) on a beat-to-beat basis via movement of cytoskeletal proteins. In translational studies we have used this response to “report” mitochondrial function in models of cardiomyopathy and to test efficacy of novel therapies to prevent cardiomyopathy.

Ion channel molecular complexes in vascular smooth muscle

Article

Full-text available

Aug 2022

Ion channels that influence membrane potential and intracellular calcium concentration control vascular smooth muscle excitability. Voltage-gated calcium channels (VGCC), transient receptor potential (TRP) channels, voltage (K V ), and Ca ²⁺ -activated K ⁺ (BK) channels are key regulators of vascular smooth muscle excitability and contractility. These channels are regulated by various signaling cues, including protein kinases and phosphatases. The effects of these ubiquitous signaling molecules often depend on the formation of macromolecular complexes that provide a platform for targeting and compartmentalizing signaling events to specific substrates. This manuscript summarizes our current understanding of specific molecular complexes involving VGCC, TRP, and K V and BK channels and their contribution to regulating vascular physiology.

Mapping molecular determinants of Ca V 2.2 inhibition by RGK proteins and homologs in Xenopus oocytes

Preprint

Full-text available

Jun 2022

The Ca V 1 and Ca V 2 families of voltage-dependent calcium channels play a crucial role in neurotransmitter release, excitation-contraction and many other cellular processes. Comprised of the membrane pore-forming α 1 , intracellular β and extracellular α 2 δ subunits, these channels have been targets for pharmacological intervention for decades. Physiological functions of Ca V channels are attenuated by either constitutively or transiently bounds proteins in the cellular environment. The RGK (Rad, Gem, Rem, and Rem2) G-protein family potently inhibits Ca V 1 and Ca V 2 function in heterologous expression systems. RGK proteins bind to Ca V β and inhibit channel localization and activity by forming a ternary complex with Ca V α 1 . Here, we evaluated the influence of RGK proteins on Ca V 2.2 channels heterologously expressed in Xenopus oocytes. Both Gem and Rad showed no nucleotide dependency on its inhibitory function on Ca V 2.2. The G-domain and C-terminus could inhibit the Ca V 2.2 channel independently when co-expressed with channel subunits. Our results demonstrated that structural determinants in Gem, crucial for channel inhibition, lie within the 222-296 amino acid region containing both the partial G-domain and C-terminus as determined from chimeric Ca V β-Gem constructs. We expanded our mapping efforts and prepared various chimeras of Drosophila melanogaster ( Dm ) RGK sequences fused to Ca V β and showed that 22 residues in RGK2t and RGK3L C-terminal imparted complete Ca V 2.2 inhibition. Point mutations in the Dm RGK C-terminus, conserved in mammalian RGK proteins, abrogated the Ca V 2.2 inhibition to a significant extent, pointing to a hot region in the extreme C-terminus for inhibition of Ca V channels. Since RGK homologs are now recognized as physiological modulators in β-adrenergic regulation of Ca V channels, the relevance of this curious G-protein family deserves close examination.

Impaired Subcortical Processing of Amplitude-Modulated Tones in Mice Deficient for Cacna2d3, a Risk Gene for Autism Spectrum Disorders in Humans

Article

Full-text available

Apr 2022

Temporal processing of complex sounds is a fundamental and complex task in hearing and a prerequisite for processing and understanding vocalization, speech, and prosody. Here, we studied response properties of neurons in the inferior colliculus (IC) in mice lacking Cacna2d3, a risk gene for autism spectrum disorders (ASDs). The α2δ3 auxiliary Ca2+ channel subunit encoded by Cacna2d3 is essential for proper function of glutamatergic synapses in the auditory brainstem. Recent evidence has shown that much of auditory feature extraction is performed in the auditory brainstem and IC, including processing of amplitude modulation (AM). We determined both spectral and temporal properties of single- and multi-unit responses in the IC of anesthetized mice. IC units of α2δ3-/- mice showed normal tuning properties yet increased spontaneous rates compared with α2δ3+/+ When stimulated with AM tones, α2δ3-/- units exhibited less precise temporal coding and reduced evoked rates to higher modulation frequencies (fm). Whereas first spike latencies (FSLs) were increased for only few modulation frequencies, population peak latencies were increased for fm ranging from 20 to 100 Hz in α2δ3-/- IC units. The loss of precision of temporal coding with increasing fm from 70 to 160 Hz was characterized using a normalized offset-corrected (Pearson-like) correlation coefficient, which appeared more appropriate than the metrics of vector strength. The processing deficits of AM sounds analyzed at the level of the IC indicate that α2δ3-/- mice exhibit a subcortical auditory processing disorder (APD). Similar deficits may be present in other mouse models for ASDs.

Mechanisms and Regulation of Cardiac CaV1.2 Trafficking

Article

Full-text available

May 2021
INT J MOL SCI

During cardiac excitation contraction coupling, the arrival of an action potential at the ventricular myocardium triggers voltage-dependent L-type Ca2+ (CaV1.2) channels in individual myocytes to open briefly. The level of this Ca2+ influx tunes the amplitude of Ca2+-induced Ca2+ release from ryanodine receptors (RyR2) on the junctional sarcoplasmic reticulum and thus the magnitude of the elevation in intracellular Ca2+ concentration and ultimately the downstream contraction. The number and activity of functional CaV1.2 channels at the t-tubule dyads dictates the amplitude of the Ca2+ influx. Trafficking of these channels and their auxiliary subunits to the cell surface is thus tightly controlled and regulated to ensure adequate sarcolemmal expression to sustain this critical process. To that end, recent discoveries have revealed the existence of internal reservoirs of preformed CaV1.2 channels that can be rapidly mobilized to enhance sarcolemmal expression in times of acute stress when hemodynamic and metabolic demand increases. In this review, we provide an overview of the current thinking on CaV1.2 channel trafficking dynamics in the heart. We highlight the numerous points of control including the biosynthetic pathway, the endosomal recycling pathway, ubiquitination, and lysosomal and proteasomal degradation pathways, and discuss the effects of β-adrenergic and angiotensin receptor signaling cascades on this process.

Skeletal Functions of Voltage Sensitive Calcium Channels

Article

Full-text available

Apr 2021
Curr Osteoporos Rep

Voltage-sensitive calcium channels (VSCCs) are ubiquitous multimeric protein complexes that are necessary for the regulation of numerous physiological processes. VSCCs regulate calcium influx and various intracellular processes including muscle contraction, neurotransmission, hormone secretion, and gene transcription, with function specificity defined by the channel’s subunits and tissue location. The functions of VSCCs in bone are often overlooked since bone is not considered an electrically excitable tissue. However, skeletal homeostasis and adaptation relies heavily on VSCCs. Inhibition or deletion of VSCCs decreases osteogenesis, impairs skeletal structure, and impedes anabolic responses to mechanical loading. Recent Findings While the functions of VSCCs in osteoclasts are less clear, VSCCs have distinct but complementary functions in osteoblasts and osteocytes. Purpose of Review This review details the structure, function, and nomenclature of VSCCs, followed by a comprehensive description of the known functions of VSCCs in bone cells and their regulation of bone development, bone formation, and mechanotransduction.

CACNA1S haploinsufficiency confers resistance to New World arenavirus infection

Article

Jul 2020

Significance It is well known that genetic differences among individuals can influence their susceptibility to infectious disease. Our results, using New World arenavirus-infected mice heterozygous for the α1S chain of the VGCC, human cells heterozygous for a mutation of this chain, and drugs that target this receptor, highlight the influence of genetic background on the outcome of infection and zoonoses. These findings also confirm the use of VGCCs as potential therapeutic targets and pave the way for understanding the relationship between genetics, susceptibility to infection, and antiviral drug efficacy.

CaV1.2 and β-Adrenergic Regulation of Cardiac Function Overview of the β-adrenergic regulation of L-type calcium channels

Book

Jan 2013

New aspects in cardiac L-type Ca2+ channel regulation

Article

Full-text available

Feb 2020

Cardiac excitation–contraction coupling is initiated with the influx of Ca2+ ions across the plasma membrane through voltage-gated L-type calcium channels. This process is tightly regulated by modulation of the channel open probability and channel localization. Protein kinase A (PKA) is found in close association with the channel and is one of the main regulators of its function. Whether this kinase is modulating the channel open probability by phosphorylation of key residues or via alternative mechanisms is unclear. This review summarizes recent findings regarding the PKA-mediated channel modulation and will highlight recently discovered regulatory mechanisms that are independent of PKA activity and involve protein–protein interactions and channel localization.

RBM20 Regulates CaV1.2 Surface Expression by Promoting Exon 9* Inclusion of CACNA1C in Neonatal Rat Cardiomyocytes

Article

Full-text available

Nov 2019
INT J MOL SCI

The CACNA1C gene encodes for the CaV1.2 protein, which is the pore subunit of cardiac l-type voltage-gated calcium (Ca2+) channels (l-channels). Through alternative splicing, CACNA1C encodes for various CaV1.2 isoforms with different electrophysiological properties. Splice variants of CaV1.2 are differentially expressed during heart development or pathologies. The molecular mechanisms of CACNA1C alternative splicing still remain incompletely understood. RNA sequencing analysis has suggested that CACNA1C is a potential target of the splicing factor RNA-binding protein motif 20 (RBM20). Here, we aimed at elucidating the role of RBM20 in the regulation of CACNA1C alternative splicing. We found that in neonatal rat cardiomyocytes (NRCMs), RBM20 overexpression promoted the inclusion of CACNA1C’s exon 9*, whereas the skipping of exon 9* occurred upon RBM20 siRNA knockdown. The splicing of other known alternative exons was not altered by RBM20. RNA immunoprecipitation suggested that RBM20 binds to introns flanking exon 9*. Functionally, in NRCMs, RBM20 overexpression decreased l-type Ca2+ currents, whereas RBM20 siRNA knockdown increased l-type Ca2+ currents. Finally, we found that RBM20 overexpression reduced CaV1.2 membrane surface expression in NRCMs. Taken together, our results suggest that RBM20 specifically regulates the inclusion of exon 9* in CACNA1C mRNA, resulting in reduced cell-surface membrane expression of l-channels in cardiomyocytes.

Presynaptic α2δ subunits are key organizers of glutamatergic synapses

Preprint

Full-text available

Oct 2019

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels (VGCCs) have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple loss-of-function model, we demonstrate a failure in presynaptic differentiation associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Mutating the MIDAS site in α2δ-2 dissociates rescuing presynaptic synapsin expression from calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. Firstly, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Secondly, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as trans-synaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

Dose-dependent and Isoform-specifi c Modulation of Ca 2+ Channels by RGK GTPases

Article

Nov 2006

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca 2+ infl ux in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be signifi cant in a physiological context. Here we show that RGKs-Gem, Rem, and Rem2-decreased Ca V 1.2 Ca 2+ current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca 2+ currents and affect Ca 2+ infl ux during action potentials. To explore how RGKs infl uence gating kinetics, we separated the roles mediated by the Ca 2+ channel accessory β subunit's interaction with its high affi nity binding site in the pore-forming α 1C subunit (AID) from its other puta-tive contact sites by utilizing an α 1C •β3 concatemer in which the AID was mutated to prevent β subunit interaction. This mutant concatemer generated currents with all the hallmarks of β subunit modulation, demonstrating that AID-β-independent interactions are suffi cient for β subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specifi c mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca 2+ channel current modulation.

Both gain‐of‐function and loss‐of‐function de novo CACNA 1A mutations cause severe developmental epileptic encephalopathies in the spectrum of Lennox‐Gastaut syndrome

Article

Aug 2019
EPILEPSIA

Objective: Developmental epileptic encephalopathies (DEEs) are genetically heterogeneous severe childhood-onset epilepsies with developmental delay or cognitive deficits. In this study, we explored the pathogenic mechanisms of DEE-associated de novo mutations in the CACNA1A gene. Methods: We studied the functional impact of four de novo DEE-associated CACNA1A mutations, including the previously described p.A713T variant and three novel variants (p.V1396M, p.G230V, and p.I1357S). Mutant cDNAs were expressed in HEK293 cells, and whole-cell voltage-clamp recordings were conducted to test the impacts on CaV 2.1 channel function. Channel localization and structure were assessed with immunofluorescence microscopy and three-dimensional (3D) modeling. Results: We find that the G230V and I1357S mutations result in loss-of-function effects with reduced whole-cell current densities and decreased channel expression at the cell membrane. By contrast, the A713T and V1396M variants resulted in gain-of-function effects with increased whole-cell currents and facilitated current activation (hyperpolarized shift). The A713T variant also resulted in slower current decay. 3D modeling predicts conformational changes favoring channel opening for A713T and V1396M. Significance: Our findings suggest that both gain-of-function and loss-of-function CACNA1A mutations are associated with similarly severe DEEs and that functional validation is required to clarify the underlying molecular mechanisms and to guide therapies.

Exploring the molecular basis of neuronal excitability in a vocal learner

Article

Full-text available

Aug 2019
BMC GENOMICS

Background: Vocal learning, the ability to learn to produce vocalizations through imitation, relies on specialized brain circuitry known in songbirds as the song system. While the connectivity and various physiological properties of this system have been characterized, the molecular genetic basis of neuronal excitability in song nuclei remains understudied. We have focused our efforts on examining voltage-gated ion channels to gain insight into electrophysiological and functional features of vocal nuclei. A previous investigation of potassium channel genes in zebra finches (Taeniopygia guttata) revealed evolutionary modifications unique to songbirds, as well as transcriptional specializations in the song system [Lovell PV, Carleton JB, Mello CV. BMC Genomics 14:470 2013]. Here, we expand this approach to sodium, calcium, and chloride channels along with their modulatory subunits using comparative genomics and gene expression analysis encompassing microarrays and in situ hybridization. Results: We found 23 sodium, 38 calcium, and 33 chloride channel genes (HGNC-based classification) in the zebra finch genome, several of which were previously unannotated. We determined 15 genes are missing relative to mammals, including several genes (CLCAs, BEST2) linked to olfactory transduction. The majority of sodium and calcium but few chloride channels showed differential expression in the song system, among them SCN8A and CACNA1E in the direct motor pathway, and CACNG4 and RYR2 in the anterior forebrain pathway. In several cases, we noted a seemingly coordinated pattern across multiple nuclei (SCN1B, SCN3B, SCN4B, CACNB4) or sparse expression (SCN1A, CACNG5, CACNA1B). Conclusion: The gene families examined are highly conserved between avian and mammalian lineages. Several cases of differential expression likely support high-frequency and burst firing in specific song nuclei, whereas cases of sparse patterns of expression may contribute to the unique electrophysiological signatures of distinct cell populations. These observations lay the groundwork for manipulations to determine how ion channels contribute to the neuronal excitability properties of vocal learning systems.

Dysfunctional Cav1.2 channel in Timothy syndrome, from cell to bedside

Article

Full-text available

Jul 2019

Timothy syndrome is a rare disorder caused by CACNA1C gene mutations and characterized by multi-organ system dysfunctions, including ventricular arrhythmias, syndactyly, dysmorphic facial features, intermittent hypoglycemia, immunodeficiency, developmental delay, and autism. Because of the low morbidity and high mortality at a young age, it remains a huge challenge to establish a diagnosis and treatment system to manage Timothy syndrome patients. Here, we aim to provide a detailed review of Timothy syndrome, discuss the mechanisms underlying dysfunctional Cav1.2 due to CACNA1C mutations, and provide some new emerging evidences in treating Timothy syndrome from cell to bedside, promoting the management of this rare disease. Impact statement The knowledge of Timothy syndrome (TS) caused by dysfunctional Cav1.2 channel due to CACNA1C mutations is rapidly evolving as novel technologies of electrophysiology are introduced and our understanding of the mechanisms of TS develops. In this review, we focus on the TS-related dysfunctional Cav1.2 and the underlying mechanisms. We update TS-related CACNA1C mutations in a precise way over the past 20 years and summarize all reported TS patients based on their clinical presentations and molecular mechanisms, respectively. We hope this review will provide a new comprehensive way to better understand the electrophysiological mechanisms underlying TS from cell to bedside, promoting the management of TS in practice.

Transcription Factor Sp1 Regulates the Expression of Calcium Channel α2δ-1 Subunit in Neuropathic Pain

Article

Jun 2019
NEUROSCIENCE

High voltage-activated (HVA) Ca2+ (CaV) channels are oligomeric complexes formed by an ion-conducting main subunit (Cavα1) and at least two auxiliary subunits (Cavβ and CaVα2δ). It has been reported that the expression of CaVα2δ1 increases in the dorsal root ganglia (DRGs) of animals with mechanical allodynia, and that the transcription factor Sp1 regulates the expression of the auxiliary subunit. Hence, the main aim of this work was to investigate the role of Sp1 as a molecular determinant of the exacerbated expression of CaVα2δ-1 in the nerve ligation-induced model of mechanical allodynia. Our results show that ligation of L5/L6 spinal nerves (SNL) produced allodynia and increased the expression of Sp1 and CaVα2δ-1 in the DRGs. Interestingly, intrathecal administration of the Sp1 inhibitor mithramycin A (Mth) prevented allodynia and decreased the expression of Sp1 and CaVα2δ-1. Likewise, electrophysiological recordings showed that incubation with Mth decreased Ca2+ current density in the DRG neurons, acting mostly on HVA channels. These results suggest that L5/L6 SNL produces mechanical allodynia and increases the expression of the transcription factor Sp1 and the subunit CaVα2δ-1 in the DRGs, while Mth decreases mechanical allodynia and Ca2+ currents through HVA channels in sensory neurons by reducing the functional expression of the CaVα2δ-1 subunit.

Nociceptor Signalling through ion Channel Regulation via GPCRs

Article

Full-text available

May 2019
INT J MOL SCI

The prime task of nociceptors is the transformation of noxious stimuli into action potentials that are propagated along the neurites of nociceptive neurons from the periphery to the spinal cord. This function of nociceptors relies on the coordinated operation of a variety of ion channels. In this review, we summarize how members of nine different families of ion channels expressed in sensory neurons contribute to nociception. Furthermore, data on 35 different types of G protein coupled receptors are presented, activation of which controls the gating of the aforementioned ion channels. These receptors are not only targeted by more than 20 separate endogenous modulators, but can also be affected by pharmacotherapeutic agents. Thereby, this review provides information on how ion channel modulation via G protein coupled receptors in nociceptors can be exploited to provide improved analgesic therapy.

Lack of correlation between the activity of the mesolimbic dopaminergic system and the rewarding properties of pregabalin in mouse

Article

Full-text available

Jul 2019
PSYCHOPHARMACOLOGY

Rationale Pregabalin is a psychoactive drug indicated in the treatment of epilepsy, neuropathic pain, and generalized anxiety disorders. Pregabalin acts on different neurotransmission systems by inactivating the alpha2-delta subunit of voltage-gated calcium channels. In light of this pharmacological property, the hypothesis has been raised that pregabalin may regulate the mesolimbic dopamine pathway and thereby display a potential for misuse or abuse as recently observed in humans. Although some preclinical data support this possibility, the rewarding properties of gabapentinoid are still a matter for debate. Objective The aim of this work was to evaluate the rewarding properties of pregabalin and to determine its putative mechanism of action in healthy mice. Results Pregabalin alone (60 mg/kg; s.c.) produced a rewarding effect in the conditioned place preference (CPP) test albeit to a lower extent than cocaine (30 mg/kg; s.c.). Interestingly, when assessing locomotor activity in the CPP, the PGB60 group, similarly to the cocaine group, showed an increased locomotor activity. In vivo single unit extracellular recording showed that pregabalin had mixed effects on dopamine (DA) neuronal activity in the ventral tegmental area since it decreased the activity of 50% of neurons and increased 28.5% of them. In contrast, cocaine decreased 75% of VTA DA neuronal activity whereas none of the neurons were activated. Intracerebal microdialysis was then conducted in awake freely mice to determine to what extent such electrophysiological parameters influence the extracellular DA concentrations ([DA]ext) in the nucleus accumbens. Although pregabalin failed to modify this parameter, cocaine produced a robust increase (800%) in [DA]ext. Conclusions Collectively, these electrophysiological and neurochemical experiments suggest that the rewarding properties of pregabalin result from a different mode of action than that observed with cocaine. Further experiments are warranted to determine whether such undesirable effects can be potentiated under pathological conditions such as neuropathic pain, mood disorders, or addiction and to identify the key neurotransmitter system involved.

Alterations in Schizophrenia-Associated Genes Can Lead to Increased Power in Delta Oscillations

Article

Full-text available

Nov 2018
CEREB CORTEX

Genome-wide association studies have implicated many ion channels in schizophrenia pathophysiology. Although the functions of these channels are relatively well characterized by single-cell studies, the contributions of common variation in these channels to neurophysiological biomarkers and symptoms of schizophrenia remain elusive. Here, using computational modeling, we show that a common biomarker of schizophrenia, namely, an increase in delta-oscillation power, may be a direct consequence of altered expression or kinetics of voltage-gated ion channels or calcium transporters. Our model of a circuit of layer V pyramidal cells highlights multiple types of schizophrenia-related variants that contribute to altered dynamics in the delta-frequency band. Moreover, our model predicts that the same membrane mechanisms that increase the layer V pyramidal cell network gain and response to delta-frequency oscillations may also cause a deficit in a single-cell correlate of the prepulse inhibition, which is a behavioral biomarker highly associated with schizophrenia.

Methyl-β-Cyclodextrin Impairs the Phosphorylation of the β2 Subunit of L-Type Calcium Channels and Cytosolic Calcium Homeostasis in Mature Cerebellar Granule Neurons

Article

Full-text available

Nov 2018
INT J MOL SCI

The activation of L-type calcium channels (LTCCs) prevents cerebellar granule neurons (CGNs) from entering low-K+-induced apoptosis. In previous works, we showed that LTCCs are largely associated with caveolin-1-rich lipid rafts in the CGN plasma membrane. In this work, we show that protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMK-II) are associated with caveolin-1-rich lipid rafts of mature CGNs, and we further show that treatment with the cholesterol-trapping and lipid raft-disrupting agent methyl-β-cyclodextrin decreases the phosphorylation level of the LTCC β2 subunit and the steady-state calcium concentration in neuronal somas ([Ca2+]i) to values close to those measured in 5 mM KCl proapoptotic conditions. These effects correlate with the effects produced by a short (15 min) treatment of CGNs with H-89 and KN-93—inhibitors of PKA and CaMK-II, respectively—in 25 mM KCl medium. Moreover, only a 15 min incubation of CGNs with H-89 produces about a 90% inhibition of the calcium entry that would normally occur through LTCCs to increase [Ca2+]i upon raising the extracellular K+ from 5 to 25 mM, i.e., from proapoptotic to survival conditions. In conclusion, the results of this work suggest that caveolin-1-rich lipid rafts play a major role in the control of the PKA- and CaMK-II-induced phosphorylation level of the LTCC β2 subunit, thus preventing CGNs from entering apoptosis.

Increased α2δ‐1–NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy‐induced neuropathic pain

Article

Full-text available

Dec 2018
J NEUROCHEM

Painful peripheral neuropathy is a severe and difficult‐to‐treat neurological complication associated with cancer chemotherapy. Although chemotherapeutic drugs such as paclitaxel are known to cause tonic activation of presynaptic NMDA receptors (NMDARs) to potentiate nociceptive input, the molecular mechanism involved in this effect is unclear. α2δ‐1, commonly known as a voltage‐activated calcium channel subunit, is a newly discovered NMDAR‐interacting protein and plays a critical role in NMDAR‐mediated synaptic plasticity. Here we show that paclitaxel treatment in rats increases the α2δ‐1 expression level in the dorsal root ganglion and spinal cord and the mRNA levels of GluN1, GluN2A, and GluN2B in the spinal cord. Paclitaxel treatment also potentiates the α2δ‐1–NMDAR interaction and synaptic trafficking in the spinal cord. Strikingly, inhibiting α2δ‐1 trafficking with pregabalin, disrupting the α2δ‐1–NMDAR interaction with an α2δ‐1 C‐terminus–interfering peptide, or α2δ‐1 genetic ablation fully reverses paclitaxel treatment‐induced presynaptic NMDAR‐mediated glutamate release from primary afferent terminals to spinal dorsal horn neurons. In addition, intrathecal injection of pregabalin or α2δ‐1 C‐terminus–interfering peptide and α2δ‐1 knockout in mice markedly attenuate paclitaxel‐induced pain hypersensitivity. Our findings indicate that α2δ‐1 is required for paclitaxel‐induced tonic activation of presynaptic NMDARs at the spinal cord level. Targeting α2δ‐1–bound NMDARs, not the physiological α2δ‐1–free NMDARs, may be a new strategy for treating chemotherapy‐induced neuropathic pain. Open science badges This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. image

An African loss-of-function CACNA1C variant p.T1787M associated with a risk of ventricular fibrillation

Article

Full-text available

Oct 2018

Calcium regulation plays a central role in cardiac function. Several variants in the calcium channel Cav1.2 have been implicated in arrhythmic syndromes. We screened patients with Brugada syndrome, short QT syndrome, early repolarisation syndrome, and idiopathic ventricular fibrillation to determine the frequency and pathogenicity of Cav1.2 variants. Cav1.2 related genes, CACNA1C, CACNB2 and CACNA2D1, were screened in 65 probands. Missense variants were introduced in the Cav1.2 alpha subunit plasmid by mutagenesis to assess their pathogenicity using patch clamp approaches. Six missense variants were identified in CACNA1C in five individuals. Five of them, A1648T, A1689T, G1795R, R1973Q, C1992F, showed no major alterations of the channel function. The sixth C-terminal variant, Cavα1c-T1787M, present mostly in the African population, was identified in two patients with resuscitated cardiac arrest. The first patient originated from Cameroon and the second was an inhabitant of La Reunion Island with idiopathic ventricular fibrillation originating from Purkinje tissues. Patch-clamp analysis revealed that Cavα1c-T1787M reduces the calcium and barium currents by increasing the auto-inhibition mediated by the C-terminal part and increases the voltage-dependent inhibition. We identified a loss-of-function variant, Cavα1c-T1787M, present in 0.8% of the African population, as a new risk factor for ventricular arrhythmia.

Design and Applications of Genetically-Encoded Voltage-Dependent Calcium Channel Inhibitors

Chapter

Jun 2023
Handbook Exp Pharmacol

Ca2+ influx through high-voltage-gated Ca2+ channels (HVGCCs; CaV1/CaV2) is an exceptionally powerful and versatile signal that controls numerous cell and physiological functions including neurotransmission, muscle contraction, and regulation of gene expression. The impressive ability of a singular signal, Ca2+ influx, to have such a plethora of functional outcomes is enabled by: molecular diversity of HVGCC pore-forming α1 and auxiliary subunits; organization of HVGCCs with extrinsic modulatory and effector protein to form discrete macromolecular complexes with unique properties; distinctive distribution of HVGCCs into separate subcellular compartments; and varying expression profiles of HVGCC isoforms among different tissues and organs. The capacity to block HVGCCs with selectivity and specificity with respect to the different levels of their organization is critical for fully understanding the scope of functional consequences of Ca2+ influx through them, and is also important for realizing their full potential as therapeutic targets. In this review, we discuss the gaps in the current landscape of small-molecule HVGCC blockers and how these may be addressed with designer genetically-encoded Ca2+ channel inhibitors (GECCIs) that draw inspiration from physiological protein inhibitors of HVGCCs.

Protein composition of axonal dopamine release sites in the striatum

Article

Full-text available

Dec 2022
eLife

Dopamine is an important modulator of cognition and movement. We recently found that evoked dopamine secretion is fast and relies on active zone-like release sites. Here, we used in vivo biotin-identification (iBioID) proximity proteomics in mouse striatum to assess which proteins are present at these sites. Using three release site baits, we identified proteins that are enriched over the general dopamine axonal protein content, and they fell into categories including active zone, Ca ²⁺ regulatory and synaptic vesicle proteins. We also detected many proteins not previously associated with vesicular exocytosis. Knockout of the presynaptic organizer protein RIM strongly decreased the hit number obtained with iBioID, while Synaptotagmin-1 knockout did not. α-Synuclein, a protein linked to Parkinson's disease, was enriched at release sites, and its enrichment was lost in both tested mutants. We conclude that RIM organizes scaffolded dopamine release sites and provide a proteomic assessment of the composition of these sites.

A family-based study of genetic and epigenetic effects across multiple neurocognitive, motor, social-cognitive and social-behavioral functions

Article

Full-text available

Dec 2022
BEHAV BRAIN FUNCT

Many psychiatric and neurodevelopmental disorders are known to be heritable, but studies trying to elucidate the genetic architecture of such traits often lag behind studies of somatic traits and diseases. The reasons as to why relatively few genome-wide significant associations have been reported for such traits have to do with the sample sizes needed for the detection of small effects, the difficulty in defining and characterizing the phenotypes, partially due to overlaps in affected underlying domains (which is especially true for cognitive phenotypes), and the complex genetic architectures of the phenotypes, which are not wholly captured in traditional case–control GWAS designs. We aimed to tackle the last two issues by performing GWASs of eight quantitative neurocognitive, motor, social-cognitive and social-behavioral traits, which may be considered endophenotypes for a variety of psychiatric and neurodevelopmental conditions, and for which we employed models capturing both general genetic association and parent-of-origin effects, in a family-based sample comprising 402 children and their parents (mostly family trios). We identified 48 genome-wide significant associations across several traits, of which 3 also survived our strict study-wide quality criteria. We additionally performed a functional annotation of implicated genes, as most of the 48 associations were with variants within protein-coding genes. In total, our study highlighted associations with five genes ( TGM3 , CACNB4 , ANKS1B, CSMD1 and SYNE1 ) associated with measures of working memory, processing speed and social behavior. Our results thus identify novel associations, including previously unreported parent-of-origin associations with relevant genes, and our top results illustrate new potential gene → endophenotype → disorder pathways.

Vascular CaV1.2 channels in diabetes

Chapter

Oct 2022
CURR TOP MEMBR

Diabetic vasculopathy is a significant cause of morbidity and mortality in the diabetic population. Hyperglycemia, one of the central metabolic abnormalities in diabetes, has been associated with vascular dysfunction due to endothelial cell damage. However, studies also point toward vascular smooth muscle as a locus for hyperglycemia-induced vascular dysfunction. Emerging evidence implicates hyperglycemia-induced regulation of vascular L-type Ca2 + channels CaV1.2 as a potential mechanism for vascular dysfunction during diabetes. This chapter summarizes our current understanding of vascular CaV1.2 channels and their regulation during physiological and hyperglycemia/diabetes conditions. We will emphasize the role of CaV1.2 in vascular smooth muscle, the effects of elevated glucose on CaV1.2 function, and the mechanisms underlying its dysregulation in hyperglycemia and diabetes. We conclude by examining future directions and gaps in knowledge regarding CaV1.2 regulation in health and during diabetes.

Strukturelle Differenzierung und Plastizität präsynaptischer Aktiver Zonen

Thesis

Full-text available

Jan 2022

Achmed Mrestani

Ziel der vorliegenden Arbeit war die nanoskopische Analyse struktureller Differenzierung und Plastizität präsynaptischer aktiver Zonen (AZs) an der NMJ von Drosophila melanogaster mittels hochauflösender, lichtmikroskopischer Bildgebung von Bruchpilot (Brp). In erster Linie wurde das lokalisationsmikroskopische Verfahren dSTORM angewendet. Es wurden neue Analyse-Algorithmen auf der Basis von HDBSCAN entwickelt, um eine objektive, in weiten Teilen automatisierte Quantifizierung bis auf Ebene der Substruktur der AZ zu ermöglichen. Die Differenzierung wurde am Beispiel phasischer und tonischer Synapsen, die an dieser NMJ durch Is- und Ib-Neurone gebildet werden, untersucht. Phasische Is-Synapsen mit hoher Freisetzungswahrscheinlichkeit zeigten kleinere, kompaktere AZs mit weniger Molekülen und höherer molekularer Dichte mit ebenfalls kleineren, kompakteren Brp-Subclustern. Akute strukturelle Plastizität wurde am Beispiel präsynaptischer Homöostase, bei der es zu einer kompensatorisch erhöhten Neurotransmitterfreisetzung kommt, analysiert. Interessanterweise zeigte sich hier ebenfalls eine kompaktere Konfiguration der AZ, die sich auch auf Ebene der Subcluster widerspiegelte, ohne Rekrutierung von Molekülen. Es konnte demonstriert werden, dass sich eine höhere Moleküldichte in der Lokalisationsmikroskopie in eine höhere Intensität und größere Fläche in der konfokalen Mikroskopie übersetzt, und damit der Zusammenhang zu scheinbar gegensätzlichen Vorbefunden hergestellt werden. Die Verdichtung bzw. Kompaktierung erscheint im Zusammenhang mit der Kopplungsdistanz zwischen VGCCs und präsynaptischen Vesikeln als plausibles Muster der effizienten Anordnung molekularer Komponenten der AZ. Die hier eingeführten Analysewerkzeuge und molekularbiologischen Strategien, basierend auf dem CRISPR/Cas9-System, zur Markierung von AZ-Komponenten können zukünftig zur weiteren Klärung der Bedeutung der molekularen Verdichtung als allgemeines Konzept der AZ-Differenzierung beitragen.

More than a pore: How voltage-gated calcium channels act on different levels of neuronal communication regulation

Article

Full-text available

Jan 2021

Voltage-gated calcium channels (VGCCs) represent key regulators of the calcium influx through the plasma membrane of excitable cells, like neurons. Activated by the depolarization of the membrane, the opening of VGCCs induces very transient and local changes in the intracellular calcium concentration, known as calcium nanodomains, that in turn trigger calcium-dependent signaling cascades and the release of chemical neurotransmitters. Based on their central importance as concierges of excitation-secretion coupling and therefore neuronal communication, VGCCs have been studied in multiple aspects of neuronal function and malfunction. However, studies on molecular interaction partners and recent progress in omics technologies have extended the actual concept of these molecules. With this review, we want to illustrate some new perspectives of VGCCs reaching beyond their function as calcium-permeable pores in the plasma membrane. Therefore, we will discuss the relevance of VGCCs as voltage sensors in functional complexes with ryanodine receptors, channel-independent actions of auxiliary VGCC subunits, and provide an insight into how VGCCs even directly participate in gene regulation. Furthermore, we will illustrate how structural changes in the intracellular C-terminus of VGCCs generated by alternative splicing events might not only affect the biophysical channel characteristics but rather determine their molecular environment and downstream signaling pathways.

Adrenergic Ca V 1.2 Activation via Rad Phosphorylation Converges at α 1C I-II Loop

Article

Oct 2020

Rationale: Changing activity of cardiac Ca V 1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently up-regulated via activation of protein kinase A, essential molecular details remained stubbornly enigmatic. Objective: The primary goal of this study was to determine how various factors converging at the Ca V 1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure. Methods and Results: We generated transgenic mice with expression of Ca V 1.2 α 1C subunits with: 1) mutations ablating interaction between α 1C and β subunits; 2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α 1C ); or 3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α 1C up-regulated in the hypertrophied heart. Introducing three glycine residues that disrupt a rigid IS6-AID helix markedly reduced basal open probability despite intact binding of Ca V β to α 1C I-II loop, and eliminated β-adrenergic agonist stimulation of Ca V 1.2 current. In contrast, introduction of the exon 9* splice variant in α 1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β 2B and Rad or transgenically expressed in cardiomyocytes. Conclusions: Ca ²⁺ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α 1C I-II loop. Ca V β binding to α 1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca ²⁺ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β binding to α 1C .

Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease

Article

Full-text available

Aug 2020

Significance Hypertrophic cardiomyopathy affects 1:500 of the general population. Current drug therapy is used to manage symptoms in patients. There is an unmet need for treatments that can prevent the cardiomyopathy. Here we identify biomarkers of hypertrophic cardiomyopathy resulting from causing cardiac troponin I mutation Gly203Ser, and present a safe, nontoxic, preventative approach for the treatment of associated cardiomyopathy.

Presynaptic calcium channels: specialized control of synaptic neurotransmitter release

Article

Mar 2020
NAT REV NEUROSCI

Chemical synapses are heterogeneous junctions formed between neurons that are specialized for the conversion of electrical impulses into the exocytotic release of neurotransmitters. Voltage-gated Ca2+ channels play a pivotal role in this process as they are the major conduits for the Ca2+ ions that trigger the fusion of neurotransmitter-containing vesicles with the presynaptic membrane. Alterations in the intrinsic function of these channels and their positioning within the active zone can profoundly alter the timing and strength of synaptic output. Advances in optical and electron microscopic imaging, structural biology and molecular techniques have facilitated recent breakthroughs in our understanding of the properties of voltage-gated Ca2+ channels that support their presynaptic functions. Here we examine the nature of these channels, how they are trafficked to and anchored within presynaptic boutons, and the mechanisms that allow them to function optimally in shaping the flow of information through neural circuits. Voltage-gated calcium channels have an essential role in the regulation of neurotransmitter release. Dolphin and Lee describe here how advances in the techniques available to study presynaptic voltage-gated calcium channels have provided insight into their composition, trafficking, regulation and contributions to presynaptic function.

Oligomerization of Ca v β subunits is an essential correlate of Ca 2+ channel activity

Article

Dec 2010

Excitation-contraction coupling in skeletal muscle: recent progress and unanswered questions

Article

Jan 2020

Dmitry Shishmarev

Excitation-contraction coupling (ECC) is a physiological process that links excitation of muscles by the nervous system to their mechanical contraction. In skeletal muscle, ECC is initiated with an action potential, generated by the somatic nervous system, which causes a depolarisation of the muscle fibre membrane (sarcolemma). This leads to a rapid change in the transmembrane potential, which is detected by the voltage-gated Ca2+ channel dihydropyridine receptor (DHPR) embedded in the sarcolemma. DHPR transmits the contractile signal to another Ca2+ channel, ryanodine receptor (RyR1), embedded in the membrane of the sarcoplasmic reticulum (SR), which releases a large amount of Ca2+ ions from the SR that initiate muscle contraction. Despite the fundamental role of ECC in skeletal muscle function of all vertebrate species, the molecular mechanism underpinning the communication between the two key proteins involved in the process (DHPR and RyR1) is still largely unknown. The goal of this work is to review the recent progress in our understanding of ECC in skeletal muscle from the point of view of the structure and interactions of proteins involved in the process, and to highlight the unanswered questions in the field.

Calcium Signaling in Neurons and Glial Cells: Role of Cav1 channels

Article

Oct 2019
NEUROSCIENCE

Calcium (Ca2+) is an essential component in intracellular signaling of brain cells, and its control mechanisms are of great interest in biological systems. Ca2+ can signal differently in neurons and glial cells using the same intracellular pathways or cell membrane structural components. These types of machinery are responsible for entry, permanence, and removal of Ca2+ from the cellular environment and are of vital importance for brain homeostasis. This review highlights the importance of Ca2+ in neuronal and glial cell physiology as well as aspects of learning, memory, and Alzheimer's disease, focusing on the involvement of L-type voltage-gated Ca2+ channels.

Clinical and functional studies of autoimmune disorders of neuromuscular transmission

Thesis

Feb 2015

Jennifer Spillane

Inherited and acquired disorders of the neuromuscular junction are an important cause of muscle weakness and fatigability. In this thesis I focus on the autoimmune disorders of neuromuscular transmission. Myasthenia Gravis (MG) is the most common of these diseases and is typically caused by antibodies against the post-synaptic acetylcholine receptor. Lambert Eaton Myasthenic Syndrome (LEMS) is a pre-synaptic disorder typically caused by antibodies against voltage gated calcium channels (VGCC). With regard to LEMS, my main aim was to gain a more complete understanding of the pathomechanisms of the disease. To date, the direct effect of LEMS IgG on presynaptic neurotransmitter release had not been investigated in detail. I examined how LEMS IgG affects neurotransmitter release by imaging action potential dependent vesicle exocytosis using a fluorescent dye. I found that LEMS IgG significantly inhibited the rate of synaptic vesicle release but this effect was lost in synapses from a Cacna1a knockout mouse. These data provide direct evidence that LEMS is caused by impaired neurotransmitter release due to an effect on P/Q-type VGCCs. With regard to MG, I studied the long-term outcome of patients with thymomatous and non-thymomatous MG after thymectomy and found that in general the outcome was favourable in the majority of patients with 34% of patients achieving complete stable remission. I also reviewed the long-term outcome of patients after a severe exacerbation of MG requiring ITU admission. Despite the significant mortality associated with severe exacerbations of MG, it was found that specialised neuro-intensive care was associated with a good long-term prognosis in the majority of patients. There were no significant differences in outcome in those with early or late onset MG. Overall the data presented in this thesis provide new insights into the pathomechanisms of LEMS IgG and provide new information regarding the long-term outcome of patients with MG.

CACNB2 is associated with aberrant RAS-MAPK signaling in hypertensive Dahl Salt-Sensitive rats

Article

Apr 2019

Several independent genome-wide association studies (GWAS) have indicated that calcium (Ca2+) voltage-gated channel auxiliary subunit beta 2 (CACNB2) an L-type Ca2+ channel (LTCC) associated protein has strong association with hypertension. However, the molecular mechanism of CACNB2 and its role in the pathophysiology of hypertension is not clear. To address this knowledge gap, we utilized in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt-Sensitive (SS) rats. We demonstrated that CACNB2 over-expression in HEK293 cells triggers cell proliferation via an up-regulation of the RAS-MAPK pathway compared to non-transfected cells. These effects were likely independent of LTCC activity as treatment with nifedipine, a well-known LTCC blocker, in CACNB2 overexpressing cells failed to inhibit the RAS-MAPK pathway gene expressions or show an effect on apoptosis marker gene expression. Furthermore, the expression level of CACNB2 was up-regulated in the high salt (HS) diet fed SS rat kidneys compared to low salt diet (LS) fed group. Similar to our in vitro observation the RAS-MAPK mRNA levels were increased in HS fed SS rat kidneys, compared to LS fed group. Collectively, our data suggest that CACNB2 is associated with the increase in RAS-MAPK gene expressions and lead us to speculate that in addition to its role in regulating LTCC α1-subunit trafficking, CACNB2 might lead to aberrant RAS activation, which is one of the key cascade associated with hypertension. KEYWORDS: CACNB2; Dahl SS rat; L-type calcium channel; RAS–MAPK

The β 4 subunit of Ca v 1.2 channels is required for an optimal interferon response in cardiac muscle cells

Article

Full-text available

Dec 2018

The auxiliary β 4 subunit of the cardiac Ca v 1.2 channel plays a poorly understood role in gene transcription. Here, we characterized the regulatory effects of the β 4 subunit in H9c2 rat cardiac cells on the abundances of Ifnb mRNA [which encodes interferon-β (IFN-β)] and of the IFN-β–related genes Ddx58 , Ifitm3 , Irf7 , Stat2 , Ifih1 , and Mx1 , as well as on the abundances of the antiviral proteins DDX58, IRF7, STAT2, and IFITM3. Knocking down the β 4 subunit in H9c2 cells reduced the expression of IFN-β–stimulated genes. In response to inhibition of the kinase JAK1, the abundances of β 4 subunit mRNA and protein were decreased. β 4 subunit abundance was increased, and it translocated to the nucleus, in cells treated with IFN-β, infected with dengue virus (DENV), or transfected with poly(I:C), a synthetic analog of double-stranded RNA. Cells that surrounded the virus-infected cells showed translocation of β 4 subunit proteins to nuclei in response to spreading infection. We showed that the β 4 subunit interacted with the transcriptional regulator IRF7 and that the activity of an Irf7 promoter–driven reporter was increased in cells overexpressing the β 4 subunit. Last, overexpressing β 4 in undifferentiated and differentiated H9c2 cells reduced DENV infection and decreased the abundance of the viral proteins NS1, NS3, and E-protein. DENV infection and poly(I:C) also increased the concentration of intracellular Ca ²⁺ in these cells. These findings suggest that the β 4 subunit plays a role in promoting the expression of IFN-related genes, thereby reducing viral infection.

Targeted disruption of the Ca2+ channel 3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltagedependent activation of P/Q-type Ca2+ channels in neurons

Article

Full-text available

Sep 1998
P NATL ACAD SCI USA

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding α1 subunit, considerably less is understood about how β subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel β3 subunit, the major Ca2+ channel β subunit in neurons, by using a gene-targeting strategy. The β3 deficient (β3−/−) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic β3−/− neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the “reluctant” and “willing” states reported for N-type channels. The absence of the β3 subunit was associated with a hyperpolarizing shift of the “reluctant” component of activation. Norepinephrine inhibited wt and β3−/− neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the β3−/− mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.

Interaction of NE-dlg/SAP102, a Neuronal and Endocrine Tissue-specific Membrane-associated Guanylate Kinase Protein, with Calmodulin and PSD-95/SAP90

Article

Full-text available

Feb 1999

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends ofN-methyl-d-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic α-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a K d value of 44 nm. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.

Unique regulatory properties of the type 2a Ca2+ channel subunit caused by palmitoylation

Article

Full-text available

Apr 1998

Beta subunits of voltage-gated Ca2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for beta2a, which differs in that it does not support prepulse facilitation of alpha1C Ca2+ channels, inhibits voltage-induced inactivation of neuronal alpha1E Ca2+ channels, and is more effective in blocking inhibition of alpha1E channels by G protein-coupled receptors. We show that the distinguishing properties of beta2a, rather than interaction with a distinct site of alpha1, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of beta2a were lost in a mutant that could not be palmitoylated [beta2a(Cys3,4Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three beta2 splice variants differing only in their N-termini.

Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single G subunit

Article

Full-text available

Mar 1998

We have examined the modulation of cloned and stably expressed rat brain N type calcium channels (alpha1B + beta1b + alpha2delta subunits) by exogenously applied purified G protein betagamma subunits. In the absence of Gbetagamma, barium currents through N type channels are unaffected by application of strong depolarizing prepulses. In contrast, inclusion of purified Gbetagamma in the patch pipette results in N type currents that initially facilitated upon application of positive prepulses followed by rapid reinhibition. Examination of the kinetics of Gbetagamma-dependent reinhibition showed that as the duration between the test pulse and the prepulse was increased, the degree of facilitation was attenuated in a monoexponential fashion. The time constant tau for the recovery from facilitation was sensitive to exogenous Gbetagamma, so that the inverse of tau linearly depended on the Gbetagamma concentration. Overall, the data are consistent with a model whereby a single Gbetagamma molecule dissociates from the channel during the prepulse, and that reassociation of Gbetagamma with the channel after the prepulse occurs as a bimolecular reaction.

Acceleration of activation and inactivation by the ?? subunit of the skeletal muscle calcium channel

Article

Full-text available

Aug 1991

The L-type voltage-dependent calcium channel is an important link in excitation-contraction coupling of muscle cells (reviewed in refs 2 and 3). The channel has two functional characteristics: calcium permeation and receptor sites for calcium antagonists. In skeletal muscle the channel is a complex of five subunits, alpha 1, alpha 2, beta, gamma and delta. Complementary DNAs to these subunits have been cloned and their amino-acid sequences deduced. The skeletal muscle alpha 1 subunit cDNA expressed in L cells manifests as specific calcium-ion permeation, as well as sensitivity to the three classes of organic calcium-channel blockers. We report here that coexpression of the alpha 1 subunit with other subunits results in significant changes in dihydropyridine binding and gating properties. The available number of drug receptor sites increases 10-fold with an alpha 1 beta combination, whereas the affinity of the dihydropyridine binding site remains unchanged. Also, the presence of the beta subunit accelerates activation and inactivation kinetics of the calcium-channel current.

Calcium channel ?? subunit heterogeneity: Functional expression of cloned cDNA from heart, aorta and brain

Article

Full-text available

Apr 1992

Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L-type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.

Cloning and expression of cardiac/brain ?? subunit of the L-type calcium channel

Article

Full-text available

Feb 1992

The skeletal muscle dihydropyridine receptor/Ca2+ channel is composed of five protein components (alpha 1, alpha 2 delta, beta, and gamma). Only two such components, alpha 1 and alpha 2, have been identified in heart. The present study reports the cloning and expression of a novel beta gene that is expressed in heart, lung, and brain. Coexpression of this beta with a cardiac alpha 1 in Xenopus oocytes causes the following changes in Ca2+ channel activity: it increases peak currents, accelerates activation kinetics, and shifts the current-voltage relationship toward more hyperpolarized potentials. It also increases dihydropyridine binding to alpha 1 in COS cells. These results indicate that the cardiac L-type Ca2+ channel has a similar subunit structure as in skeletal muscle, and provides evidence for the modulatory role of the beta subunit.

Skeletal muscle and brain isoform of a ?? subunit of human voltage-dependent calcium channels are encoded by a single gene

Article

Full-text available

Dec 1992

Clones of the beta 1-subunit of the voltage-dependent calcium channel (VDCC) from human skeletal muscle and hippocampus cDNA libraries, and from human genomic libraries, were isolated using a human skeletal muscle beta 1 cDNA probe generated by polymerase chain reaction. The skeletal muscle beta 1 cDNA (beta 1M) encodes a protein of 523 amino acids that is 97% identical to the rabbit skeletal muscle beta-subunit. Two different cDNAs, beta 1B1 and beta 1B2, were obtained from the human hippocampus library. The beta 1B1 transcript encodes a protein of 478 amino acids that is identical to the skeletal muscle beta-subunit (beta 1M), except for an internal region of 52 amino acids. The beta 1B2 transcript encodes a protein of 596 amino acids. The beta 1B2 polypeptide is identical to the beta 1B1 polypeptide at amino acids 1-444; however, it has a unique 152 amino acid carboxyl terminus. Like beta 1B1, it differs from beta 1M at the internal 52 amino acids. Analysis of the beta 1 gene structure demonstrates that these three cDNAs represent transcripts encoded by a single beta 1 gene. Transcripts from the beta 1 gene were detected in RNA from skeletal muscle, heart, spleen, and brain, but not in RNA from liver, stomach, or kidney.

The roles of subunits in the function of the calcium channel

Article

Full-text available

Oct 1991

Dihydropyridine-sensitive voltage-dependent L-type calcium channels are critical to excitation-secretion and excitation-contraction coupling. The channel molecule is a complex of the main, pore-forming subunit alpha 1 and four additional subunits: alpha 2, delta, beta, and gamma (alpha 2 and delta are encoded by a single messenger RNA). The alpha 1 subunit messenger RNA alone directs expression of functional calcium channels in Xenopus oocytes, and coexpression of the alpha 2/delta and beta subunits enhances the amplitude of the current. The alpha 2, delta, and gamma subunits also have pronounced effects on its macroscopic characteristics, such as kinetics, voltage dependence of activation and inactivation, and enhancement by a dihydropyridine agonist. In some cases, specific modulatory functions can be assigned to individual subunits, whereas in other cases the different subunits appear to act in concert to modulate the properties of the channel.

Large depolarization induces long openings of voltage-dependent calcium channels in adrenal chromaffin cells

Article

Full-text available

Mar 1987

Single Ca2+-channel currents in bovine adrenal chromaffin cells were studied with the patch-clamp technique using Ba2+ as the charge carrier. Depolarizing pulses to voltages less than +10 mV from holding voltage of -60 mV elicited short openings with a mean life time of less than 1 msec. Depolarization to more positive voltages elicited longer openings with a mean life time of about 3 msec in addition to the short openings similar to those observed at less positive voltages. Following large depolarizing prepulses, 2 types of "tail" openings, one with a mean duration of less than 1 msec and the other with a mean duration of 4 msec, were observed. In the presence of a dihydropyridine BAY K 8644, openings with a mean duration of more than 12 msec were present. Depolarization-induced long openings and BAY K 8644-produced long openings differed in the first latency and open-time properties. The results could be explained in terms of multiple open states of one type of Ca2+ channel. A kinetic model with at least 2 open states is required to explain activation of Ca2+ channels in chromaffin cells.

Subunit structure of DHP-sensitive calcium channels from skeletal muscle

Article

Full-text available

Sep 1987

Purified dihydropyridine-sensitive calcium channels from rabbit transverse-tubule membranes consist of three noncovalently associated classes of subunits: alpha (167 kDa), beta (54 kDa), and gamma (30 kDa). Cleavage of disulfide bonds reveals two distinct alpha polypeptides and an additional component, delta. The alpha 1 subunit, a 175-kDa polypeptide that is not N-glycosylated, contains the dihydropyridine binding site, cAMP-dependent protein kinase phosphorylation site(s), and substantial hydrophobic domain(s). alpha 2, a 143-kDa glycoprotein, has none of the properties characteristic of alpha 1 but binds lectins and contains about 25% N-linked carbohydrate. alpha 2 is disulfide-linked to delta, a 24- to 27-kDa glycopeptide. beta (54 kDa) contains a cAMP-dependent phosphorylation site but is not N-glycosylated and does not have a hydrophobic domain. gamma (30 kDa) has a carbohydrate content of about 30% and extensive hydrophobic domain(s). Precipitation with affinity-purified anti-alpha 1 antibodies or alpha 2-specific lentil lectin-agarose demonstrated that alpha 1 alpha 2 beta gamma delta behaves as a complex in the presence of digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, whereas the alpha 2 delta complex dissociates from alpha 1 beta gamma in the presence of Triton X-100. A model for subunit interaction and membrane insertion is proposed on the basis of these observations.

cAMP-dependent protein kinase rapidly phosphorylates serine- 687 of the skeletal muscle receptor for calcium channel blockers

Article

Full-text available

Nov 1988

The cAMP-dependent phosphorylation of the 165-kDa subunit of the receptor for organic calcium channel blockers (CaCB-receptors) was studied. Tryptic peptide analysis showed that cAMP-dependent protein kinase phosphorylates rapidly a serine in one peptide. Up to three peptides containing phosphoserine and -threonine are phosphorylated in a 2-h incubation. The isolated 165-kDa subunit was digested with trypsin and the endoproteinase Lys-C and Glu-C. The rapidly phosphorylated peptide was isolated from each digest. The amino acid sequence was determined by Edman degradation and compared with the deduced amino acid sequence of the CaCB-receptor from rabbit skeletal muscle (Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T., and Numa, S. (1987) Nature 238, 313-318). Phosphoserine was determined as the phenylthiohydantoin-derivative of dithiothreitol-dehydroalanine. The phosphorylated serine was identified as Ser-687 which is localized between the transmembrane regions II and III. A second phosphopeptide was isolated into which phosphate was incorporated into Ser-1617 with a slow time course. This peptide is located in the COOH-terminal cytoplasmic domain of the 165-kDa subunit. It is anticipated that phosphorylation of serine 687 affects the opening probability of the calcium channel.

Dihydropyridine and phenylalkylamine receptors associated with cardiac and skeletal muscle calcium channels are structurally different

Article

Full-text available

Jan 1989

We have purified putative L-type Ca2+ channels from chick heart by virtue of their associated high affinity receptors for the Ca2+ channel effectors, dihydropyridines (DHPs), and phenylalkylamines (PAAs). A peptide of 185,000-190,000 daltons was found to comigrate with the peak of DHP binding activity during purification through two successive cycles of lectin affinity chromatography and sucrose density gradient centrifugation. A previously described peptide of 140,000 daltons, whose Mr was increased to approximately 180,000 under nonreducing conditions, also copurified with the 185-kDa peptide and dihydropyridine binding activity. When cardiac membranes were photolabeled with either the dihydropyridine [3H]azidopine or the PAA [3H]azidopamil prior to purification, a single, specifically labeled component of 185,000-190,000 daltons was present in the purified fractions. The properties of this 185-kDa cardiac DHP/PAA receptor were compared to the smaller 165-kDa DHP/PAA receptor previously purified from skeletal muscle. Antibodies raised against the 165-kDa skeletal muscle DHP/PAA receptor reacted with both rabbit and chick skeletal muscle receptors, but only poorly recognized, if at all, the cardiac 185-190 kDa component. The 185-kDa peptide present in the purified fractions obtained from cardiac muscle did not undergo substantial phosphorylation by cAMP-dependent protein kinase, while the purified 165-kDa peptide from rabbit and chick skeletal muscle was a good substrate for this kinase. The results show that the DHP and PAA receptors in cardiac muscle are contained in a 185-190-kDa peptide that is significantly larger than, and structurally and immunologically different from, it skeletal muscle counterpart.

Immunochemical identification and subcellular distribution of the ??1A subunits of brain calcium channels

Article

Full-text available

Nov 1995

A site-directed anti-peptide antibody (anti-CNA1) directed against the alpha 1 subunit of class A calcium channels (alpha 1A) recognized a protein of approximately 190-200 kDa in immunoblot and immunoprecipitation analyses of rat brain glycoproteins. Calcium channels recognized by anti-CNA1 were distributed throughout the brain with a high concentration in the cerebellum. Calcium channels having alpha 1A subunits were concentrated in presynaptic terminals making synapses on cell bodies and on dendritic shafts and spines of many classes of neurons and were especially prominent in the synapses of the parallel fibers of cerebellar granule cells on Purkinje neurons where their localization in presynaptic terminals was confirmed by double labeling with the synaptic membrane protein syntaxin or the microinjected postsynaptic marker Neurobiotin. They were present in lower density in the surface membrane of dendrites of most major classes of neurons. There was substantial labeling of Purkinje cell bodies, but less intense staining of the cell bodies of hippocampal pyramidal neurons, layer V pyramidal neurons in the dorsal cortex, and most other classes of neurons in the forebrain and cerebellum. Scattered cell bodies elsewhere in the brain were labeled at low levels. These results define a unique pattern of localization of class A calcium channels in the cell bodies, dendrites, and presynaptic terminals of most central neurons. Compared to class B N-type calcium channels, class A calcium channels are concentrated in a larger number of presynaptic nerve terminals implying a more prominent role in neurotransmitter release at many central synapses.

Calcium channel β subunit binds to a consered motif in the I–II cytoplasmic linker of the α1 subunit

Article

Full-text available

Apr 1994

The beta-subunit is an integral component of purified voltage-sensitive Ca2+ channels. Modulation of Ca2+ channel activity by the beta-subunit, which includes significant increases in transmembrane current and/or changes in kinetics, is observed on coexpression of six alpha 1-subunit genes with four beta-subunit genes in all alpha 1-beta combinations tested. Recent reports suggest that this regulation is not due to targeting of the alpha 1-subunit to the plasma membrane but is probably a result of a conformational change induced by the beta-subunit. Here we report that the beta-subunit binds to the cytoplasmic linker between repeats I and II of the dihydropyridine-sensitive alpha 1-subunits from skeletal (alpha 1S) and cardiac muscles (alpha 1C-a), and also with the more distantly related neuronal alpha 1A and omega-conotoxin GVIA-sensitive alpha 1B-subunits. Sequence analysis of the beta-subunit binding site identifies a conserved motif (QQ-E--L-GY--WI--E) positioned 24 amino acids from the IS6 transmembrane domain in each alpha 1-subunit. Mutations within this motif reduce the stimulation of peak currents by the beta-subunit and alter inactivation kinetics and voltage-dependence of activation. Conservation of the beta-subunit binding motif in these functionally distinct calcium channels suggests a critical role for the I-II cytoplasmic linker of the alpha 1-subunit in channel modulation by the beta-subunit.

Chimeric L-type Ca2+ channels expressed in Xenopus laevis oocytes reveal role of repeats III and IV in activation gating

Article

Full-text available

Aug 1995

1. Chimeric alpha 1 subunits consisting of repeat I and II from the rabbit cardiac (alpha 1C-a) and repeat III and IV from the carp skeletal muscle Ca2+ channel (alpha 1S) were constructed and expressed in Xenopus laevis oocytes without co-expressing other channel subunits. Ba2+-current kinetics of five chimeric channel constructs were studied in Xenopus oocytes using the two-microelectrode technique. 2. Exchange of repeats III and IV of alpha 1C-a with sequences of alpha 1S results in a significantly slower and biexponential activation (apparent activation time constants tau 1act = 19.8 +/- 1.8 ms and tau 2act = 214 +/- 28.7 ms, n = 7) of expressed Ca2+ channel currents; no current inactivation was observable during an 800 ms test pulse to 0 mV. 3. Activation of a chimera consisting of repeats I, II and IV from the alpha 1C-a subunit and repeat III from alpha 1S was fast and monoexponential (tau 1act = 6.33 +/- 1.7 ms, n = 5) and the current inactivated during a 350 ms test pulse to 0 mV (tau inact = 175 +/- 22 ms, n = 5). The current kinetics of this construct did not significantly differ from kinetics of a construct consisting of repeats I to IV from alpha 1C-a (tau 1act = 6.6 +/- 2.1 ms; tau inact = 198 +/- 14 ms; n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Association of Native Ca2+ Channel β Subunits with the α1 Subunit Interaction Domain

Article

Full-text available

Aug 1995

β Subunits of voltage-dependent Ca channels play an important role in regulating Ca channel function. The sites of α1-β subunit interaction have been localized recently to cytoplasmic domains of both subunits. The α1 subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all α1 subunits which is essential for the binding of β subunits. In order to further study the interaction of β subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the α subunit that contains the AID. Mutant GST fusion proteins that contain a single amino acid change (Y392S, Y392F, and Y392W) in the AIDA along with control GST were coupled to glutathione-Sepharose beads to form affinity beads. Binding assays using these affinity beads with in vitro synthesized S-labeled β2 and β3 subunits demonstrate that the hydroxyl group on tyrosine 392 of AIDA is critical for binding to β subunits. The affinity bead assay was also used to identify and characterize native β subunits from detergent extracts of different tissues. The AIDA affinity beads, but not the control or Y392S beads, specifically bind β subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of β in skeletal muscle, β2 and β3 in cardiac muscle, and β, β3, and β4 in brain. The assays also demonstrate the AIDA beads bind to β subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of β subunits which is not always associated with α1 subunits. Also, β subunits from solubilized skeletal muscle triads can be affinity-purified using AIDA CNBr-Sepharose. Our data demonstrate that the AID binds to native β subunits from detergent and non-detergent tissue extracts illustrating that this domain on the α1 subunit is the major anchoring site for the β subunit.

Cloning and expression of a neuronal calcium channel β subunit

Article

Full-text available

Jul 1993

Although pharmacological and electrophysiological studies have demonstrated the existence of multiple types of voltage-dependent calcium channels in neuronal tissue, the subunit composition of these channels is not well known. Here, we report the cloning and expression of a new rat brain beta subunit (beta 4). Northern blot analysis indicates that beta 4 mRNA is expressed almost exclusively in neuronal tissues, with the highest levels being found in the cerebellum. Coexpression studies indicate that rat beta 4 can interact with rabbit cardiac muscle alpha 1, rabbit skeletal muscle alpha 1, and calcium channels endogenous to Xenopus oocytes. beta 4 modulation of alpha 1 activity is similar to the modulation induced by beta 1, beta 2, or beta 3. The most striking effect of beta subunits is their ability to increase functional alpha 1 activity, which can be measured as either increased dihydropyridine binding to membranes from transfected COS cells or increased calcium channel activity in Xenopus oocytes.

Voltage-dependent potentiation of the activity of cardiac L-type calcium channel α1 subunits due to phosphorylation by cAMP-dependent protein kinase

Article

Full-text available

Dec 1993

Barium currents mediated by the alpha 1 subunit of the cardiac L-type Ca channel expressed in Chinese hamster ovary (CHO) cells were increased up to 10-fold during dialysis of the cell with the catalytic subunit of cAMP-dependent protein kinase. After partial activation by exogenous kinase, the activity of the alpha 1 subunit was also reversibly potentiated up to 3.5-fold by prepulses to voltages in the range of 0 to +150 mV. Potentiation at +48 mV developed with a biphasic time course with time constants of 131 ms and 8 s. Reversal at -60 mV was biphasic with half-times of 12 ms and 100 ms and was blocked in the presence of the phosphatase inhibitor okadaic acid. Both the increase in calcium-channel activity during dialysis with kinase and the voltage-dependent potentiation were accompanied by shifts in the voltage dependence of activation to more negative membrane potentials. The increases in Ba current due to protein phosphorylation and to the dihydropyridine Ca channel agonist Bay K8644 were approximately additive. The results show that the alpha 1 subunit of the cardiac L-type Ca channel is sufficient for substantial modulation of Ca-channel activity by cAMP-dependent protein kinase and for potentiation by state-dependent protein phosphorylation. Voltage-dependent potentiation of the activity of the alpha 1 subunit may contribute to the increase in contractile force in response to increased rate of stimulation, the positive staircase effect in heart muscle.

Properties of the - Anchoring Site in Voltage-dependent Ca Channels

Article

Full-text available

May 1995

In voltage-dependent Ca channels, the β subunit interacts with the α1 subunit via a cytoplasmic site. A biochemical assay has been developed to quantitatively describe the interaction between both subunits. In vitro synthesized S-labeled β subunits specifically bind to a glutathione S -transferase (GST) fusion protein containing the α interaction domain (AID, located between the amino-acids 383 and 400 of the cytoplasmic loop between the hydrophobic domains I and II). Kinetic analysis demonstrates that the association of S-labeled β subunit to the AID GST fusion protein occurs with a fast rate constant at 4°C. The binding is almost irreversible as demonstrated by the absence of dissociation observed after an 8-h incubation with an 18-amino acid synthetic AID peptide. The α-β binding site does not seem to be a target for cytoplasmic regulation. The interaction is mostly unaffected by changes in ionic strength, pH, and Ca concentration or by protein kinase C phosphorylation. The specificity of subunit interaction in voltage-dependent Ca channels was also followed by saturation analyses. The data obtained show that the AID GST fusion protein binds to a single site on the β with an apparent Kof 5 nM. The affinities of AID GST fusion protein for various β subunits was measured and demonstrate that β subunits associate with different affinities to each α interaction domain. The rank order of AID affinity for each β subunit is as follows: β > β > β β. The binding of the β subunit to α subunit can be inhibited in vitro by the AID synthetic peptide with an apparent Kof 285 nM. This interaction can also be prevented in heterologous Ca channels by the injection of the AID GST fusion protein into Xenopus oocytes. Our results demonstrate that the site of interaction between AID and β subunit is responsible for anchoring the β subunit to the α subunit and thus allowing the β subunit to modify Ca channel activity.

Voltage-dependent facilitation of a neuronal ??1C L-type calcium channel

Article

Full-text available

Dec 1994

Calcium entry into excitable cells through voltage-gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non-L-type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage-dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage-dependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity.

PostTranslational Modifications of β Subunits of Voltage-Dependent Calcium Channels

Article

Aug 1998

Different post-translational modifications of Ca channel ß subunits have been identified. Recent studies have characterized the palmitoylation of the Ca channel ß2a subunit, as well as one effect of this modification on channel function. The potential importance of palmitoylation on other channel properties is discussed. Other studies have addressed the role of phosphorylation of ß subunits in the regulation of voltage-dependent Ca channels. Phosphorylation of ß subunits by second messenger-activated protein kinases, as well as by unidentified protein kinases, may affect interactions between channel subunits and other aspects of channel function. The differential modification of Ca channel ß subunit isoforms by post-translational events likely results in diversely regulated channels with unique properties.

Structure of the SH3-Guanylate Kinase Module from PSD95 Suggests a Mechanism for Regulated Assembly of MAGUK Scaffolding Proteins

Article

Dec 2001
MOL CELL

Membrane-associated guanylate kinases (MAGUKs), such as PSD-95, are modular scaffolds that organize signaling complexes at synapses and other cell junctions. MAGUKs contain PDZ domains, which recruit signaling proteins, as well as a Src homology 3 (SH3) and a guanylate kinase-like (GK) domain, implicated in scaffold oligomerization. The crystal structure of the SH3-GK module from PSD-95 reveals that these domains form an integrated unit: the SH3 fold comprises noncontiguous sequence elements divided by a hinge region and the GK domain. These elements compose two subdomains that can assemble in either an intra- or intermolecular fashion to complete the SH3 fold. We propose a model for MAGUK oligomerization in which complementary SH3 subdomains associate by 3D domain swapping. This model provides a possible mechanism for ligand regulation of oligomerization.

Modulation of the smooth-muscle L-type Ca2+ channel α1 subunit (α1C-b) by the β2a subunit: A peptide which inhibits binding of β to the I-II linker of α1 induces functional uncoupling

Article

Jun 2000

Modulation of the smooth-muscle Ca2+ channel α1C-b subunit by the auxiliary β2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the α1-β interaction in functional channels is sensitive to an 18-amino-acid synthetic peptide that corresponds to the sequence of the defined major interaction domain in the cytoplasmic I-II linker of α1C (AID-peptide). Ca2+ channels derived by co-expression of α1C-b and β2a subunits exhibited an about 3-fold higher open probability (P(o)) than α1C-b channels. High-P(o) gating of α1C-b·β2a channels was associated with the occurrence of long-lasting channel openings [mean open time (τ) > 10 ms] which were rarely observed in α1C-b channels. Modulation of fast gating by the β2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to β2 subunits of native Ca2+ channels. Binding of the β2 protein to immobilized AID-peptide was specifically inhibited (K1 of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 μM) to the cytoplasmic side of inside-out patches induced a substantial reduction of P(o) of α1C-b·β2a channels. The scrambled control peptide failed to affect α1C-b·β2a channels, and the AID-peptide (10 μM) did not modify α1C-b channel function in the absence of expressed β2a subunit. Our results demonstrate that the β2a subunit controls fast gating of α1C-b channels, and suggest the α1-β interaction domain in the cytoplasmic I-II linker of α1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of α1-β interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the α1-β interaction.

Mutation of the Ca 2+ Channel ?? Subunit Gene Cchb4 Is Associated with Ataxia and Seizures in the Lethargic ( lh) Mouse

Article

Feb 1997
CELL

Ca2+ channel β subunits regulate voltage-dependent calcium currents through direct interaction with α1 subunits. The β- and α1-binding motifs are conserved, and all β subunits can stimulate current amplitude, voltage dependence, and kinetics when coexpressed with various α1 subunits. We used a positional candidate approach to determine that the ataxia and seizures in the lethargic (lh) mouse arise from mutation of the β-subunit gene Cchb4 on mouse chromosome 2. A four-nucleotide insertion into a splice donor site results in exon skipping, translational frameshift, and protein truncation with loss of the α1-binding site. The lethargic phenotype is the first example of a mammalian neurological disease caused by an inherited defect in a non-pore-forming subunit of a voltage-gated ion channel.

Identification of critical amino acids involved in α 1- β interaction in voltage-dependent Ca 2+ channels

Article

Feb 1996

In voltage-dependent Ca2+ channels, the α1 and β subunits interact via two cytoplasmic regions defined as the Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AIDA GST fusion protein for the four interacting β1b BID mutants was not significantly altered compared with the wild-type β1b despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive β mutants with the full-length α1A subunit only slightly modified the stimulation efficiency when compared with the wild-type β1b subunit. Our data suggest that non-disruptive BID sequence alterations do not dramatically affect the β subunit-induced current stimulation.

Assignment of human genes for β2 and β4 subunits of voltage-dependent Ca 2+ channels to chromosomes 10p12 and 2q22-q23

Article

Jul 1997

We have used human β2 and β4 cDNA probes to map the genes encoding two isoforms of the regulatory β subunit of voltage-activated Ca2+ channels, viz. CACNB2 (β2) and CACNB4 (β4), to human chromosomes 10p12 and 2q22-q23, respectively, by fluorescence in situ hybridization. The gene encoding the β2 protein, first described as a Lambert-Eaton myasthenic syndrome (LEMS) antigen in humans, is found close to a region that undergoes chromosome rearrangements in small cell lung cancer, which occurs in association with LEMS. CACNB2 (β2) and CACNB4 (β4) genes are members of the ion-channel gene superfamily and it should now be possible to examine their loci by linkage analysis of ion-channel-related disorders. To date, no such disease-related gene has been assigned to 10p12 and 2q22-q23.

Allosteric modulation of Ca2+ channels by G proteins, voltage-dependent facilitation, protein kinase C, and CaVβ subunits

Article

Apr 2001

N-type and P/Q-type Ca(2+) channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membrane-delimited pathway involving Gbetagamma subunits. Inhibition is caused by a shift from an easily activated "willing" (W) state to a more-difficult-to-activate "reluctant" (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca(2+) channels containing Cav2.2a alpha(1) subunits and P/Q-type Ca(2+) channels containing Ca(v)2.1 alpha(1) subunits revealed substantial differences. In the absence of G protein modulation, Ca(v)2.1 channels containing Ca(v)beta subunits were tonically in the W state, whereas Ca(v)2.1 channels without beta subunits and Ca(v)2.2a channels with beta subunits were tonically in the R state. Both Ca(v)2.1 and Ca(v)2.2a channels could be shifted back toward the W state by strong depolarization or PKC phosphorylation. Our results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Ca(v)beta subunits are intrinsic properties of the Ca(2+) channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca(2+)-channel activity incorporating an intrinsic equilibrium between the W and R states of the alpha(1) subunits and modulation of that equilibrium by G proteins, Ca(v)beta subunits, membrane depolarization, and phosphorylation by PKC accommodates our findings. Such regulation will modulate transmission at synapses that use N-type and P/Q-type Ca(2+) channels to initiate neurotransmitter release.

Modulation Of Ca2+ channels by G-protein beta gamma subunits

Article

Mar 1996
NATURE

Calcium ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+ currents. This pathway regulates the activity of N-type and P/Q-type Ca2+ channels, which are localized in presynaptic terminals and participate in neurotransmitter release. Synaptic transmission is inhibited by neurotransmitters through this mechanism. G-protein alpha subunits confer specificity in receptor coupling, but it is not known whether the G alpha or G beta gamma subunits are responsible for modulation of Ca2+ channels. Here we report that G beta gamma subunits can modulate Ca2+ channels. Transfection of G beta gamma into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but G alpha subunits do not. Similarly, injection or expression of G beta gamma subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but G alpha subunits do not. In both cases, the G gamma subunit is ineffective by itself, but overexpression of exogenous G beta subunits is sufficient to cause channel modulation.

Molecular basis for K ATP assembly: transmembrane interaction s mediate associatation of a K + channel with and ABC transporter

Article

Apr 2000

K(ATP) channels are large heteromultimeric complexes containing four subunits from the inwardly rectifying K+ channel family (Kir6.2) and four regulatory sulphonylurea receptor subunits from the ATP-binding cassette (ABC) transporter family (SUR1 and SUR2A/B). The molecular basis for interactions between these two unrelated protein families is poorly understood. Using novel trafficking-based interaction assays, coimmunoprecipitation, and current measurements, we show that the first transmembrane segment (M1) and the N terminus of Kir6.2 are involved in K(ATP) assembly and gating. Additionally, the transmembrane domains, but not the nucleotide-binding domains, of SUR1 are required for interaction with Kir6.2. The identification of specific transmembrane interactions involved in K(ATP) assembly may provide a clue as to how ABC proteins that transport hydrophobic substrates evolved to regulate other membrane proteins.

The effect of 2 - and other accessory subunits on expression and properties of the calcium channel 1G

Article

Sep 2004
J PHYSIOL-LONDON

• The effect has been examined of the accessory α2- and  subunits on the properties of α1G currents expressed in monkey COS-7 cells and Xenopus oocytes. • In immunocytochemical experiments, the co-expression of α2- increased plasma membrane localization of expressed α1G and conversely, the heterologous expression of α1G increased immunostaining for endogenous α2-, suggesting an interaction between the two subunits. • Heterologous expression of α2- together with α1G in COS-7 cells increased the amplitude of expressed α1G currents by about 2-fold. This finding was confirmed in the Xenopus oocyte expression system. The truncated  construct did not increase α1G current amplitude, or increase its plasma membrane expression. This indicates that it is the exofacial α2 domain that is involved in the enhancement by α2-. • 1b also produced an increase of functional expression of α1G, either in the absence or the presence of heterologously expressed α2-, whereas the other  subunits had much smaller effects. • None of the accessory subunits had any marked influence on the voltage dependence or kinetics of the expressed α1G currents. These results therefore suggest that α2- and 1b interact with α1G to increase trafficking of, or stabilize, functional α1G channels expressed at the plasma membrane.

Calcium channels: Structure, function, and classification

Article

Nov 1994
DRUG DEVELOP RES

Voltage-gated Ca2+ channels have been extensively characterized in terms of their electrophysiological and pharmacological properties [McDonald et al. (1994): Physiol Rev 74:365–507; Spedding and Paoletti (1992): Pharmacol Rev 44:363–376; Tsien and Tsien (1990): Annu Rev Cell Biol 6:715–760]. These studies indicate that there are numerous types of Ca2+ channels, termed L, N, P/Q, R, and T [Zhang et al. (1993): Neuropharmacology 32:1075–1088]. Biochemical and molecular biological studies have established that Ca2+ channels are multi-subunit complexes composed of an ion-conducting subunit, α1 (see Fig. 1), and smaller accessory subunits (α2, β, and sometimes γ and a 95 kDa protein). To date (May, 1994), genes for six α1, four β, one α2, and one γ have been cloned. Expression studies with cloned α1 have demonstrated that this subunit can determine the voltage and pharmacological sensitivity of the channel. This should allow us to classify the cloned α1s in terms of their type. Unfortunately life is not that simple. We will review how the accessory subunits are capable of modifying the pharmacological and biophysical characteristics of the channel. Despite these complications, 5 of the 6 α1s can be classified as follows: (1) three α1s (α1s, α1c, and α1D) belong to the L-type (dihydropyridine-sensitive), (2) α1B is an N-type (ω-conotoxin-GVIA-sensitive), and (3) α1A is a P (ω-aga-IVA-sensitive, also called Q [see Zhang et al. (1993): Neuro-pharmacology 32:1075–1088], herein referred to as P/Q). The sixth α1, α1E, does not display any distinctive pharmacology, thus it has been called an R-type (resistant). The molecular biology of Ca2+ channels has its origins in the biochemical characterization of the skeletal muscle dihydropyridine receptor. This receptor/channel complex was purified, sequenced, cloned, and expressed. Cloning of these cDNAs provided the probes to discover the molecular diversity of Ca2+ channels. We will review the cloning, tissue distribution, and functional expression of α1 subunits following a historical path, then review the accessory subunits. © 1994 Wiley-Liss, Inc.

Importance of the Different |β Subunits in the Membrane Expression of the α1A and α2 Calcium Channel Subunits: Studies Using a Depolarization‐sensitive α1A Antibody

Article

Apr 1997
EUR J NEUROSCI

The plasma membrane expression of the rat brain calcium channel subunits α1A, α2-Δ and the β subunits β1b, β2a, β3b and β4 was examined by transient expression in COS-7 cells. Neither α1A nor α2-Δ localized to the plasma membrane, either alone or when coexpressed. However, coexpression of α1A or α2-Δ/α1A with any of the p subunits caused α1A and α2 to be targetted to the plasma membrane. The α1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native α1A channels in dorsal root ganglion neurons and for α1A expressed with a β subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of α1A. Immunostaining for α1A was obtained in depolarized non-permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a subunit. In contrast, β1b and β2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target α1A to the membrane since β3 and β4 alone showed no differential localization, but did direct the translocation of α1A to the plasma membrane, suggesting a chaperone role for the β subunits

Spatial and Syndromic Surveillance for Public Health

Article

Jun 1991

Continual surveillance to detect some event is of interest in quite different situations in industry, medicine, economics and other fields. A general method with an optimality property is described. The implications of the method for some different situations are derived. Some commonly used methods turn out to be special cases.

P-Type calcium channels in rat central and peripheral neurons

Article

Aug 1992
NEURON

The peptide toxin omega-Aga-IVA blocked P-type Ca2+ channel current in rat Purkinje neurons (KD approximately 2 nM) but had no effect on identified T-type, L-type, or N-type currents in a variety of central and peripheral neurons. omega-Aga-IVA blocked a substantial fraction of high threshold Ca2+ channel current in neurons from the hippocampal CA1 region (mean 26%), visual cortex (32%), spinal cord (45%), and dorsal root ganglia (23%), but less in hippocampal CA3 neurons (14%) and none in sympathetic neurons. In all cases, omega-Aga-IVA block could be reversed by a brief train of strong depolarizations. There was no overlap between current blocked by omega-Aga-IVA and the fractions blocked by dihydropyridines and omega-conotoxin GVIA, but not all current resistant to dihydropyridines and omega-conotoxin was blocked by omega-Aga-IVA. The results suggest that omega-Aga-IVA is highly selective for P-type channels and that many central neurons and some peripheral neurons possess substantial P-type current.

Modulation of calcium current gating in frog skeletal muscle by conditioning depolarization

Article

Dec 1992

1. Ca2+ inward currents were measured by voltage clamping cut skeletal muscle fibres of the frog (Rana esculenta) in a double-Vaseline-gap system. 2. In order to study the basis of the previously described fast gating mode induced in the Ca2+ inward current by a conditioning depolarization we quantitatively analysed the response to differing features of the conditioning prepulse. 3. The faster activation seen during the second of two depolarizations was confined to the component of the inward current which could be blocked by 5 to 10 microM nifedipine. 4. By applying depolarizing conditioning pulses of gradually increasing length the time course of the transition to the fast gating mode could be determined. 5. Both the transition to the fast gating mode (point 4) caused by a depolarization and the slow inward current activated during the same depolarization showed similar voltage-dependent kinetics. 6. The kinetic change of the test current appeared to be equal when the same fractional activation was achieved at the end of the conditioning pulse independent of its duration or amplitude. 7. Flash photolysis of nifedipine in the interval between conditioning and test pulse showed that the predepolarization causes a rate-enhancing effect even though the slow channels were blocked by nifedipine during the conditioning pulse. 8. We conclude that the transition of the calcium channel from its slow to its fast gating mode is determined by the slow voltage-dependent reaction which limits the rate of channel opening under control conditions. This reaction is apparently not prevented by the binding of nifedipine and the block of current flow through the channel.

Polarity signals

Article

Oct 1991
CELL

Colin R. Hopkins

Novel mechanism of voltage-dependent gating in L-type calcium channels

Article

Sep 1990

Activation of voltage-dependent calcium channels by membrane depolarization triggers a variety of key cellular responses, such as contraction in heart and smooth muscle and exocytotic secretion in endocrine and nerve cells. Modulation of calcium channel gating is believed to be the mechanism by which several neurotransmitters, hormones and therapeutic agents mediate their effects on cell function. Here we describe a novel type of voltage-dependent equilibrium between different gating patterns of dihydropyridine-sensitive (L-type) cardiac Ca2+ channels. Strong depolarizations drive the channel from its normal gating pattern into a mode of gating characterized by long openings and high open probability. The rate constants for conversions between gating modes, estimated from single channel recordings, are much slower than normal channel opening and closing rates, but the equilibrium between modes is almost as steeply voltage-dependent as channel activation and deactivation at more negative potentials. This new mechanism of voltage-dependent gating can explain previous reports of activity-dependent Ca2+ channel potentiation in cardiac and other cells and forms a potent mechanism by which Ca2+ uptake into cells could be regulated.

L‐Type Calcium Channels in Cardiac and Skeletal Muscle

Article

Feb 1989

Primary Structure of the ?? Subunit of the DHP-Sensitive Calcium Channel from Skeletal Muscle

Article

Oct 1989

Complementary DNAs for the beta subunit of the dihydropyridine-sensitive calcium channel of rabbit skeletal muscle were isolated on the basis of peptide sequences derived from the purified protein. The deduced primary structure is without homology to other known protein sequences and is consistent with the beta subunit being a peripheral membrane protein associated with the cytoplasmic aspect of the sarcolemma. The protein contains sites that might be expected to be preferentially phosphorylated by protein kinase C and guanosine 3',5'-monophosphate-dependent protein kinase. A messenger RNA for this protein appears to be expressed in brain.

Subunits of purified calcium channels: A 212-kDa form of α1 and partial amino acid sequence of a phosphorylation site of an independent β subunit

Article

Dec 1989

Antibodies prepared against peptides CP2, CP4, and CP5, which occur within the first 1522 amino acid residues of the alpha 1 subunit of dihydropyridine-sensitive skeletal muscle calcium channels, specifically recognized a 175-kDa form of the alpha 1 subunit in immunoblots and immunoprecipitation experiments. In contrast, antibodies prepared against peptide CP1, which represents the C-terminal 18 amino acid residues predicted by cloning and sequence analysis of the alpha 1 subunit, recognized a minor, previously undescribed 212-kDa protein, which is the size predicted for the full length of the alpha 1 subunit from cDNA cloning [Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1987) Nature (London) 328, 313-318]. Both the 175-kDa and 212-kDa forms were phosphorylated by cAMP-dependent protein kinase and both were present in isolated transverse tubule membranes. The 175-kDa form may arise from posttranslational proteolytic cleavage of the C terminus of the 212-kDa form of the alpha 1 subunit predicted by cDNA cloning and sequence analysis. Partial amino acid sequencing of the 54-kDa beta subunit of the calcium channel indicated this protein was not derived from the proteolytically cleaved C terminus of the alpha 1 subunit. This analysis identified a threonine residue in the sequence (Lys/Arg)-Arg-Pro-Thr-Pro of the beta subunit that was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of this residue in the beta subunit may play a role in modulation of calcium channel function. Separate functional roles of the 175-kDa form of the alpha 1 subunit in excitation-contraction coupling and of the 212-kDa form in ion conductance are proposed.

Primary structure of the receptor for calcium channel blockers from skeletal muscle

Article

Jul 1987

The complete amino-acid sequence of the receptor for dihydropyridine calcium channel blockers from rabbit skeletal muscle is predicted by cloning and sequence analysis of DNA complementary to its messenger RNA. Structural and sequence similarities to the voltage-dependent sodium channel suggest that in the transverse tubule membrane of skeletal muscle the dihydropyridine receptor may act both as voltage sensor in excitation-contraction coupling and as a calcium channel.

Sodium and calcium channels in bovine chromaffin cells

Article

Nov 1982

1. Inward currents in chromaffin cells were studied with the patch‐clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The intracellular solution contained 120 mM‐Cs ⁺ and 20 mM‐tetraethylammonium (TEA ⁺ ). Na ⁺ currents were studied after blockade of Ca ²⁺ channels with 1 mM‐Co ²⁺ applied externally. Ca ²⁺ currents were recorded after eliminating Na ⁺ currents with tetrodotoxin (TTX). The current recordings were obtained in cell‐attached, outside‐out and whole‐cell recording configurations (Hamill et al. 1981). 2. Single channel measurements gave an elementary current amplitude of 1 pA at ‐10 mV for Na ⁺ channels. This amplitude increased with hyperpolarization between ‐10 and ‐40 mV, but did not vary significantly between ‐40 and ‐70 mV. 3. The mean Na ⁺ channel open time was 1 ms at ‐30 mV. This open time decreased both with depolarization and hyperpolarization. Its value was close to the time constant of inactivation, τ h , above ‐20 mV. 4. Ensemble fluctuation analysis of Na ⁺ currents gave results consistent with those of single channel measurements. Noise power spectra obtained between ‐35 mV and 0 mV could be fitted with a single Lorentzian. A range of Na ⁺ channel densities of 1·5‐10 channels per μm ² was calculated. 5. Cell‐attached single Ca ²⁺ channel recordings were obtained in isotonic BaCl 2 solution. The single channel amplitude was 0·9 pA at ‐5 mV, and it became smaller for positive potential values. 6. At ‐5 mV, single Ba ²⁺ currents appeared as bursts of 1·9 ms mean duration containing on the average 0·6 short gaps. The burst duration was larger at positive potentials. 7. Ensemble fluctuation analysis of Ca ²⁺ channels was performed on whole‐cell recordings in external solutions containing isotonic BaCl 2 or external Ca ²⁺ (Ca o ) concentrations of 1 and 5 mM. The unit amplitude calculated in the former case was similar to that obtained in single channel measurements. 8. Noise power spectra of Ca ²⁺ or Ba ²⁺ currents could be fitted by the sum of two, but not one, Lorentzian components. 9. Tail currents could be fitted by the sum of two exponential components. The corresponding time constants had values close to those obtained with noise analysis. 10. The rising phase of Ca ²⁺ and Ba ²⁺ currents was sigmoid. It could be fitted by the sum of three exponentials. The time constant of the largest amplitude component, τ 1 , was similar to the time constants of the slow component observed in noise and tail experiments. This time constant also corresponded to the burst duration obtained in single channel measurements. 11. The value of τ 1 was larger in 5 mM‐Ca o and in isotonic Ba ²⁺ than in 5 mM‐Ba o . Thus, the kinetic properties of Ca ²⁺ channels depend on the nature and concentration of the permeating ion. 12. A simple kinetic scheme is proposed to model the activation pathway of Ca ²⁺ channels. 13. Currents in 1 mM‐Ca o and 5 mM‐Ca o showed clear reversals around +53 mV and +64 mV respectively. The outward currents observed above these potentials are most probably due to Cs ⁺ ions flowing through Ca ²⁺ channels. 14. The instantaneous current—voltage relation was obtained from tail current data in the range ‐70 to +100 mV in 5 mM‐Ca o . The resulting curve displayed an inflexion point around the reversal potential. 15. Very little inactivation of Ca ²⁺ currents was observed. However, a slow current decline was observed in some cells above +10 mV. 16. Conditioning prepulses to positive potentials had potentiating or depressing effects on Ca ²⁺ currents depending on whether the test pulse lay below or above the maximal current potential. The potentiating effect may be linked to the slowest component of the current rise observed below +10 mV. The depressing effect may be related to the slow decline obtained above +10 mV. 17. Analysis of ensemble variance and of tail current amplitudes suggested that the opening probability of Ca ²⁺ channels was at least 0·9 above +40 mV. 18. A slow rundown of Ca ²⁺ currents was observed in whole‐cell recordings. The speed of the rundown was dependent on intracellular Ca ²⁺ concentration. The rundown was apparently due to a progressive elimination of the channels available for activation. 19. The density of Ca ²⁺ channels (before rundown) was estimated at 5‐15/μm ² . 20. In cell‐attached experiments, inward current channels were often seen to follow action potentials. These events did not appear to be the usual Na ⁺ and Ca ²⁺ currents. They were probably due to cation influx of either Na ⁺ or Ba ²⁺ , depending on the pipette solution, through Ca ²⁺ ‐dependent channels. Voltage‐independent single channel activity observed in whole‐cell and outside‐out recordings may be due to the same channels.

The involvement of multiple calcium channel sub-types in glutamate release from cerebellar granule cells and its modulation by GABAB receptor activation

Article

Oct 1995
NEUROSCIENCE

In this study, we have examined both the ability of various Ca²⁺ channel sub-types to support the release of [³H]glutamate from cerebellar granule neurons and the mechanism of action involved in the modulation of glutamate release by the GABAB receptor agonist, (−)-baclofen. Cerebellar granule neurons were stimulated to release newly synthesized [³H]glutamate by K⁺-evoked depolarization. Stimulated release was entirely calcium-dependent and abolished by the presence of 200 μM cadmium. Release of glutamate was not affected by either tetrodotoxin or 5-aminophosphonovaleric acid but was potentiated by dihydrokainate and inhibited by 6-cyano-7-nitroquinoxaline-2, 3-dione. Stimulated glutamate release was partially inhibited by both the L-type calcium channel blocker, nicardipine, and the N-type calcium channel blocker, ω-conotoxin GVIA; however, the P/Q-type calcium channel blocker ω-agatoxin IVA inhibited release of glutamate only after pre-incubation of cells with ω-conotoxin GVIA.

The block of the expressed L-type calcium channel is modulated by the beta 3 subunit

Article

Nov 1995

The alpha 1C subunit of the L-type calcium channel was stable, expressed alone or in combination with the beta 3 subunit in Chinese hamster ovary cells. The beta 3 subunit enhanced significantly the inactivation of barium currents indicating that both subunits interacted with each other. The beta 3 subunit decreased significantly the half-maximal inhibitory concentration of the calcium channel blockers (-)-gallopamil and verapamil, but did not affect significantly the block caused by isradipine and mibefradil at the holding potentials of -80 mV and -40 mV. These results suggest that the beta 3 subunit affects distinctly the interaction of the expressed alpha 1C subunit with different classes of organic calcium channel blockers.

Cloning and expression of a neuronal calcium channel Β subunit

Article

Mar 1993

The skeletal muscle dihydropyridine receptor/Ca2+ channel consists of five distinct subunits (alpha 1, alpha 2 delta, beta 1, and gamma). Homologous alpha 1, alpha 2 delta, and beta 2 subunits are expressed in heart and brain. The present study reports the cloning and expression of a third beta subunit, beta 3, which is expressed predominantly in brain. Its open reading frame encodes a protein with 484 amino acids with a predicted molecular mass of 54,571 Da. Coexpression of beta 3 with a cardiac alpha 1 in Xenopus oocytes induces similar changes in Ca2+ channel activity as beta 1 and beta 2, that is, it increases peak currents, modulates the voltage dependence of activation, and accelerates activation. In addition, beta 3 accelerates the rate of inactivation at positive test potentials.

Expression of the L-type calcium channel with two different ? subunits and its modulation by Ro 40-5967

Article

Feb 1995

The smooth muscle alpha 1Cb subunit of the L-type calcium channel was expressed alone (CHO alpha 1 cell) or together with the skeletal beta 1 (CHO alpha 1 beta 1 cell) subunit or smooth muscle beta 3 (CHO alpha 1 beta 3 cell) subunit in Chinese hamster ovary (CHO) cells. The interaction of the expressed calcium channel with the non-dihydropyridine calcium channel blocker Ro 40-5967 was studied. Ro 40-5967 decreased isradipine binding by an apparent allosteric interaction and blocked the barium inward currents (IBa) in a voltage- and use-dependent manner in all cells. The steady-state inactivation curves were shifted to hyperpolarizing potentials in the presence of Ro 40-5967. The rate of channel inactivation was increased in CHO alpha 1 and CHO alpha 1 beta 3 cells. The shift in the steady-state inactivation curve and the increase in channel inactivation were less pronounced in CHO alpha 1 beta 1 cells than in the other cell lines. Low concentrations of Ro 40-5967 increased IBa by up to 198% in 33% of the CHO alpha 1 beta 1 cells. In addition, higher concentrations of Ro 40-5967 were required to inhibit IBa in 60% of the CHO alpha 1 beta 3 cells. These results suggest that the beta subunits modify the interaction of the non-dihydropyridine Ro 40-5967 with the expressed calcium channel alpha 1 subunit.

Expression of low-voltage activated Ca2+ channels from rat brain neurones in Xenopus oocytes

Article

Nov 1994
NEUROREPORT

Xenopus laevis oocytes were injected with total mRNA obtained from the thalamo-hypothalamic complex of adult rats. In 19 out of 32 injected oocytes a Ba2+ current was expressed after 4 days which could be activated at depolarizations to -70 mV from a holding potential of -120 mV and reached a maximum value at between -30 and -20 mV. The current inactivated monoexponentially with a time constant of about 420 +/- 10 ms (n = 6); its steady state inactivation had a half value of -78 +/- 1 mV (n = 8) and a slope (k) of 11.5 +/- 3.0. These characteristics are typical of LVA (T-type) Ca2+ channels in neurones from the corresponding brain structures, except for the much slower time course of inactivation. These currents were blocked by pharmacological antagonists specific for LVA channels (amiloride, flunarizine), but remained resistant to omega-Aga-IVA and omega-Cg-toxin. These results show that LVA Ca2+ channels can be expressed in oocytes provided that the corresponding mRNA is taken from brain neurones in which they are naturally well expressed.

Nucleotide bindind by the synapse associated protein SAP90

Article

Mar 1995

The rat synapse associated protein SAP90 is a member of a superfamily of potential guanylate kinases localized at cell-cell contact sites. This superfamily includes the synapse associated protein SAP97, a close relative of SAP90, the Drosophila tumor suppressor gene product dlg-Ap, the mammalian zonula occludens proteins ZO-1 and ZO-2 and the erythrocyte protein p55. Here we show that SAP90 specifically binds GMP in the micromolar range while binding to ATP, GDP and ADP is at a much lower affinity (10-25 mM), whether or not binding is detected for other guanine and adenine nucleotides. No guanylate kinase activity of SAP90 was detected under our experimental conditions. The importance of the GMP binding capacity per se and an evolutionary role for conserving of the guanylate kinase domain in this superfamily are discussed.

Multiple calcium channel types control synaptic transmission in the hippocampus

Article

Dec 1993
NEURON

N-type calcium channels play a dominant role in controlling synaptic transmission in many peripheral neurons. Transmitter release from mammalian central nerve terminals, however, is relatively resistant to the N channel antagonist omega-conotoxin GVIA. We studied the sensitivity of glutamatergic synaptic transmission in rat hippocampal slices to omega-conotoxin and to omega-Aga-IVA, a P channel antagonist. Both toxins reduced the amplitude of excitatory postsynaptic potentials in CA1 pyramidal neurons, but omega-Aga-IVA was the more rapid and efficacious. These results were corroborated by biochemical studies measuring subsecond, calcium-dependent [3H]glutamate release from hippocampal synaptosomes. Thus, at least two calcium channel types trigger glutamate release from hippocampal neurons, but P-type plays a more prominent role. Eliminating synaptic transmission in the CNS, therefore, may require inhibiting more than a single calcium channel type.

Subunits of Voltage-Gated Calcium Channels

Abstract

No full-text available

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