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Novel pharmacological modulation of dystonic phenotypes caused by a gain-of-function mutation in the Na+ leak-current channel
Abstract
The Na leak-current channel (NALCN) regulates the resting membrane potential in excitable cells, thus determining the likelihood of depolarization in response to incoming signals. Gain-of-function (gf) mutations in this channel are associated with severe dystonic movement disorders in man. Currently, there are no known pharmacological antagonists or selective modulators of this important channel. A gain-of-function mutation in NALCN of C. elegans [known as unc-77(e625)] causes uncoordinated, hyperactive locomotion. We hypothesized that this hyperactive phenotype can be rescued with pharmacological modulators. Here, we summarize the results of targeted drug screening aimed at identification of drugs that corrected locomotion deficits in unc-77(e625) animals. To assay hyperactive locomotion, animals were acutely removed from food and characteristic foraging movements were quantified. Drug screening revealed that 2-aminoethoxydiphenyl borate (2-ABP), nifedipine, nimodipine, flunarizine and ethoxzolamide significantly decreased abnormal movements in unc-77(e625) animals. 2-APB also corrected egg release and coiling deficits in this strain. In addition, serotonin and dopamine both reduced hyperactive locomotion, consistent with regulatory interactions between these systems and the NALCN. 2-APB induced movement phenotypes in wild-type animals that faithfully mimicked those observed in NALCN knockout strains, which suggested that this drug may directly block the channel. Moreover, 2-APB and flunarizine showed significant structural similarities suggestive of overlap in their mode of action. Together, these studies have revealed new insights into regulation of NALCN function and led to the discovery of a potential pharmacological antagonist of the NALCN.
... To date, there were no efficient treatments for CLIFAHDD, except for symptomatic treatments, such as clonazepam for seizures (24), acetazolamide for episodic ataxia (16), and surgical procedures for arthrogryposis (27). A 2aminoethoxydiphenyl borate and a flunarizine could rescue the hyperactive phenotype of C. elegans caused by NALCN gainof-function variants or it might serve as a direct blocker of the channel (28). However, further validation was needed for clinical implementation. ...
Background
The NALCN encodes a sodium ion leak channel that regulates nerve-resting conductance and excitability. NALCN variants are associated with two neurodevelopmental disorders, one is CLIFAHDD (autosomal dominant congenital contractures of the limbs and face, hypotonia, and developmental delay, OMIM #616266) and another is IHPRF (infantile hypotonia with psychomotor retardation, and characteristic facies 1, OMIM #615419).
Case Presentation
In the current study, a Chinese infant that manifested abnormal facial features, adducted thumbs, and neurodevelopmental retardation was diagnosed with CLIFAHDD syndrome. A trio-based whole-exome sequencing revealed that the infant harbored a de novo variant of the NALCN gene (c.4300A>G, p.I1434V).
Conclusions
Our findings further enriched the variant spectrum of the NALCN gene and may expand the clinical range of NALCN -related disorders.
... The blue asterisk on FAM155B (which co-localizes NALCN to the ER) indicates its potential involvement in X-linked dystonia parkinsonism based on its location on the X chromosome near a major risk locus. SYNJ1 and SNCA are key regulators of both SV recycling and ER function, whereas SH3GL2 refers to endophilin A, known to interact with the NALCN via SYNJ1 NALCN(lf) worms also displayed deficits in appetite and foraging (Kasap et al. 2019) plus defective insulin signaling (Dwyer and Aamodt 2013). Mice with global homozygous knockout of Nalcn typically died within 24 h after birth due to respiratory failure (Lu et al. 2007), so the effects of gene deletion on locomotion were not examined in this species. ...
Parkinson's disease (PD) is a debilitating movement disorder often accompanied by neuropsychiatric symptoms that stem from the loss of dopaminergic function in the basal ganglia and altered neurotransmission more generally. Akinesia, postural instability, tremors and frozen gait constitute the major motor disturbances, whereas neuropsychiatric symptoms include altered circadian rhythms, disordered sleep, depression, psychosis and cognitive impairment. Evidence is emerging that the motor and neuropsychiatric symptoms may share etiologic factors. Calcium/ion channels (CACNA1C, NALCN), synaptic proteins (SYNJ1) and neuronal RNA-binding proteins (RBFOX1) are among the risk genes that are common to PD and various psychiatric disorders. The Na + leak-current channel (NALCN) is the focus of this review because it has been implicated in dystonia, regulation of movement, cognitive impairment, sleep and circadian rhythms. It regulates the resting membrane potential in neurons, mediates pace-making activity, participates in synaptic vesicle recycling and is functionally co-localized to the endoplasmic reticulum (ER)-several of the major processes adversely affected in PD. Here, we summarize the literature on mechanisms and pathways that connect the motor and neuropsychiatric symptoms of PD with a focus on recurring relationships to the NALCN. It is hoped that the various connections outlined here will stimulate further discussion, suggest additional areas for exploration and ultimately inspire novel treatment strategies.
... Because the only difference between the two strains is the location of the gf mutation, these findings suggested that nimodipine was unable to bind and affect channel activity due to the nature of the mutation in the n495 allele. Nimodipine did not obviously affect locomotion in wild-type N2 animals at the same concentrations tested here (Kasap et al., 2020). ...
Two-pore domain K⁺ channels (K2 Ps) regulate the resting membrane potential in excitable cells and determine ease of depolarization. Gain-of-function (gf) mutations in one of these channels (unc-58) in C. elegans switch it to a Na⁺ conductance channel and cause tremors, paralysis and other defects. We hypothesized that it should be possible to identify drugs that corrected these defects in unc-58(gf) mutant animals by blocking or modulating the over-active channels. We examined dispersal of animals on food because the absence of effective forward locomotion is the most obvious defect. In addition, we quantified egg release over 24 hr. Starting with a known inhibitor of mammalian K2 Ps and directed structure-based screening, we evaluated numerous drugs in these assays. Loratadine, which inhibits human KCNK18, significantly improved movement as did methiothepin. We confirmed that endosulfan, a GABA-A receptor antagonist, corrected locomotion in the unc-58(gf) strains. Based on structural similarities to other hits, we found that clozapine, loxapine and amoxapine potently suppressed abnormal phenotypes. Curiously, nimodipine, a Ca⁺⁺-channel blocker, dramatically improved movement and egg laying in unc-58(e665), but not unc-58(n495) animals. Molecular modeling provided initial insights into a possible basis for this difference based on the location of the e665 and n495 mutations. This research may lead to identification of novel K2 P modulators and potential leads for drug discovery.
Cell excitability and its modulation by hormones and neurotransmitters involve the concerted action of membrane proteins, especially ion channels. Unique complements of co-expressed ion channels are exquisitely balanced against each other in different excitable cell types, establishing distinct electrical properties that are tailored for diverse physiological contributions, and dysfunction of any component may induce a disease state. A crucial parameter controlling cell excitability is the resting membrane potential (RMP) set by extra- and intra-cellular concentrations of ions, mainly Na+, K+, and Cl-, and their passive permeation across the cell membrane through leak ion channels. Indeed, dysregulation of RMP causes significant effects on cellular excitability. This review describes the molecular and physiological properties of the Na+ leak channel NALCN, which associates with its accessory subunits UNC-79, UNC-80, and NLF-1/FAM155 to conduct depolarizing background Na+ currents in various excitable cell types, especially neurons. Studies of animal models clearly demonstrate that NALCN contributes to fundamental physiological processes in the nervous system including the control of respiratory rhythm, circadian rhythm, sleep and locomotor behavior. Furthermore, dysfunction of NALCN and its subunits is associated with severe pathological states in humans. The critical involvement of NALCN in physiology is now well established, but its study has been hampered by the lack of specific drugs that can block or agonize NALCN currents in vitro and in vivo. Molecular tools and animal models are now available to accelerate our understanding of how NALCN contributes to key physiological functions, and the development of novel therapies for NALCN channelopathies.
Objectives:
Bipolar disorder (BPD) is a highly heritable psychiatric disorder whose genetic complexity and pathogenetic mechanisms are still being unraveled. The main goal of this work was to characterize BPD risk-gene candidates (identified by Nurnberger et al., JAMA Psychiatry 71:657, 2014 and Stahl et al., Nat. Genet. 51:793, 2019) with respect to their evolutionary conservation, associated phenotypes and extent of gene-gene interactions.
Methods:
Database searches and BLAST were used to identify homologous counterparts of human BPD risk genes in C. elegans, zebrafish and Drosophila. Phenotypes associated with the C. elegans genes were annotated and searched. With GeneMANIA, we characterized and quantified gene-gene interactions among members of the BPD gene set in comparison to randomly chosen gene sets of the same size.
Results:
BPD risk genes are highly conserved across species and are enriched for essential genes and genes associated with lethality and altered life span. They are significantly more interactive with each other in comparison to random genes. We identified syntenic blocks of risk genes, which provided potential insights into molecular pathways and co-morbidities associated with BPD including coronary disease, obesity and decreased life expectancy.
Conclusions:
BPD risk genes appear to be special in terms of their degree of conservation, interconnectedness and pleiotropic effects that extend beyond a role in brain function. Key hub genes or pleiotropic regulatory components may represent attractive targets for future drug discovery.
Persistently depolarizing sodium (Na+) leak currents that enhance electrical excitability have been described for decades1,2. The entity responsible for the major background Na+ conductance in neurons had remained a mystery until characterization of NALCN (Na+leak channel, non-selective)3,4. NALCN-mediated currents regulate neuronal excitability linked to respiration, locomotion and circadian rhythm4–10. NALCN activity is under tight regulation11–14 and NALCN mutations cause severe neurological disorders and early death15,16. NALCN is an orphan channel in humans, and fundamental aspects of channel assembly, gating, ion selectivity and pharmacology remain obscure. Here, we investigate this essential leak channel and determined the NALCN structure in complex with FAM155A (Family with sequence similarity 155, member A). FAM155A forms an extracellular dome that shields the ion selectivity filter from neurotoxin attack. The pharmacology of NALCN is further delineated by a walled-off central cavity with occluded lateral pore fenestrations. Clues to the modulation of NALCN activity are revealed by unusual voltage-sensor domains with asymmetric linkages to the pore. We discover a tightly closed pore gate where the vast majority of missense patient mutations cause gain-of-function phenotypes that cluster around the S6-gate and distinctive π-bulges. Our study provides a framework to demystify the physiology of NALCN and a foundation to discover treatments for NALCN channelopathies and other electrical disorders.
Introduction. Carbonic anhydrase inhibitors (CAIs) have been in clinical use for decades for the management of various disorders. An update on their drug-drug interactions is presented here considering these main therapeutic areas and drugs: glaucoma (acetazolamide, methazolamide, dichlorophenamide, dorzolamide and brinzolamide); epilepsy/obesity (sulthiame, topiramate and zonisamide); arthritis/inflammation (celecoxib and polmacoxib) and hypoxic tumors (SLC-0111).
Areas covered. Drug interactions reported between CAIs with various other pharmacological agents are reviewed in publications after 2016, when the previous review was published. Most reported interactions concern the antiepileptics sulthiame, topiramate and zonisamide, as they are part of complex regimens in which drugs controlling this disorder are administered. Fewer interactions were reported for the anti-glaucoma agents, whereas synergistic combinations of celecoxib with antibiotics or SLC-0111 with various antitumor drugs were extensively investigated.
Expert opinion. Drug interactions involving CAIs may be used both for monitoring the clinical efficacy of these agents when co-administered with other drugs but also for better controlling some diseases, such as hypoxic tumors, case for which the combination of CAIs with other anticancer agents (histone deacetylase inhibitors, alkylating agents, antimetabolite nucleosides, angiogenesis inhibitors and immune check point inhibitors) proved to be synergistic.
The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.
The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq/Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.
Objective:
To perform genotype-phenotype analysis in an infant with congenital arthrogryposis due to a de novo missense mutation in the NALCN ion channel and explore the mechanism of pathogenicity using a Caenorhabditis elegans model.
Methods:
We performed whole-exome sequencing in a preterm neonate with congenital arthrogryposis and a severe life-threatening clinical course. We examined the mechanism of pathogenicity of the associated NALCN mutation by engineering the orthologous mutation into the nematode C elegans using CRISPR-Cas9.
Results:
We identified a de novo missense mutation in NALCN, c.1768C>T, in an infant with a severe neonatal lethal form of the recently characterized CLIFAHDD syndrome (congenital contractures of the limbs and face with hypotonia and developmental delay). We report novel phenotypic features including prolonged episodes of stimulus-sensitive sustained muscular contraction associated with life-threatening episodes of desaturation and autonomic instability, extending the severity of previously described phenotypes associated with mutations in NALCN. When engineered into the C elegans ortholog, this mutation results in a severe gain-of-function phenotype, with hypercontraction and uncoordinated movement. We engineered 6 additional CLIFAHDD syndrome mutations into C elegans and the mechanism of action could be divided into 2 categories: half phenocopied gain-of-function mutants and half phenocopied loss-of-function mutants.
Conclusions:
The clinical phenotype of our patient and electrophysiologic studies show sustained muscular contraction in response to transient sensory stimuli. In C elegans, this mutation causes neuronal hyperactivity via a gain-of-function NALCN ion channel. Testing human variants of NALCN in C elegans demonstrates that CLIFAHDD can be caused by dominant loss- or gain-of-function mutations in ion channel function.
Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease. © 2015 The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected]
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Objective:
The authors sought to demonstrate that schizophrenia is a heterogeneous group of heritable disorders caused by different genotypic networks that cause distinct clinical syndromes.
Method:
In a large genome-wide association study of cases with schizophrenia and controls, the authors first identified sets of interacting single-nucleotide polymorphisms (SNPs) that cluster within particular individuals (SNP sets) regardless of clinical status. Second, they examined the risk of schizophrenia for each SNP set and tested replicability in two independent samples. Third, they identified genotypic networks composed of SNP sets sharing SNPs or subjects. Fourth, they identified sets of distinct clinical features that cluster in particular cases (phenotypic sets or clinical syndromes) without regard for their genetic background. Fifth, they tested whether SNP sets were associated with distinct phenotypic sets in a replicable manner across the three studies.
Results:
The authors identified 42 SNP sets associated with a 70% or greater risk of schizophrenia, and confirmed 34 (81%) or more with similar high risk of schizophrenia in two independent samples. Seventeen networks of SNP sets did not share any SNP or subject. These disjoint genotypic networks were associated with distinct gene products and clinical syndromes (i.e., the schizophrenias) varying in symptoms and severity. Associations between genotypic networks and clinical syndromes were complex, showing multifinality and equifinality. The interactive networks explained the risk of schizophrenia more than the average effects of all SNPs (24%).
Conclusions:
Schizophrenia is a group of heritable disorders caused by a moderate number of separate genotypic networks associated with several distinct clinical syndromes.
Ion channels are crucial components of cellular excitability and are involved in many neurological diseases. This review focuses on the sodium leak, G protein-coupled receptors (GPCRs)-activated NALCN channel that is predominantly expressed in neurons where it regulates the resting membrane potential and neuronal excitability. NALCN is part of a complex that includes not only GPCRs, but also UNC-79, UNC-80, NLF-1 and src family of Tyrosine kinases (SFKs). There is growing evidence that the NALCN channelosome critically regulates its ion conduction. Both in mammals and invertebrates, animal models revealed an involvement in many processes such as locomotor behaviors, sensitivity to volatile anesthetics, and respiratory rhythms. There is also evidence that alteration in this NALCN channelosome can cause a wide variety of diseases. Indeed, mutations in the NALCN gene were identified in Infantile Neuroaxonal Dystrophy (INAD) patients, as well as in patients with an Autosomal Recessive Syndrome with severe hypotonia, speech impairment, and cognitive delay. Deletions in NALCN gene were also reported in diseases such as 13q syndrome. In addition, genes encoding NALCN, NLF- 1, UNC-79, and UNC-80 proteins may be susceptibility loci for several diseases including bipolar disorder, schizophrenia, Alzheimer's disease, autism, epilepsy, alcoholism, cardiac diseases and cancer. Although the physiological role of the NALCN channelosome is poorly understood, its involvement in human diseases should foster interest for drug development in the near future. Toward this goal, we review here the current knowledge on the NALCN channelosome in physiology and diseases.
Using low-cost automated tracking microscopes, we have generated a behavioral database for 305 Caenorhabditis elegans strains, including 76 mutants with no previously described phenotype. The growing database currently consists of 9,203 short videos segmented to extract behavior and morphology features, and these videos and feature data are available online for further analysis. The database also includes summary statistics for 702 measures with statistical comparisons to wild-type controls so that phenotypes can be identified and understood by users.
Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-, G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ, intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly, treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs, lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (p<0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture.
How drugs bind to their receptors--from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or "pose"--has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the β(1)- and β(2)-adrenergic receptors, initially making contact with a vestibule on each receptor's extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug-receptor binding and unbinding rates.
The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na(+) (I(Leak-Na)). The I(Leak-Na) contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na(+) leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals.
Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.—Bootman, M. D., Collins, T. J., Mackenzie, L., Roderick, H. L., Berridge, M. J., Peppiatt, C. M. 2-Aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release.
Clozapine has superior and unique effects as an antipsychotic agent, but the mediators of these effects are not known. We studied behavioral and developmental effects of clozapine in Caenorhabditis elegans, as a model system to identify previously undiscovered mechanisms of drug action. Clozapine induced early larval arrest, a phenotype that was also seen with the clozapine metabolite N-desmethyl clozapine but not with any other typical or atypical antipsychotic drug tested. Mutations in the insulin receptor/daf-2 and phosphatidyl inositol 3-kinase (PI3K)/age-1 suppressed clozapine-induced larval arrest, suggesting that clozapine may activate the insulin-signaling pathway. Consistent with this notion, clozapine also increased the expression of an age-1::GFP reporter. Activation of the insulin-signaling pathway leads to cytoplasmic localization of the fork head transcription factor FOXO/daf-16. Clozapine produced cytoplasmic localization of DAF-16::GFP in arrested L1 larvae, in contrast to stressors such as starvation or high temperature, which produce nuclear localization of DAF-16::GFP in arrested L1 larvae. Clozapine also inhibited pharyngeal pumping in C. elegans, an effect that may contribute to, but did not explain, clozapine-induced larval arrest. Our findings demonstrate a drug-specific interaction between clozapine and the PI3K/insulin-signaling pathway in C. elegans. As this pathway is conserved across species, the results may have implications for understanding the unique effects of clozapine in humans.
Brainstem serotonin (5-HT) neurons modulate activity of many neural circuits in the mammalian brain, but in many cases endogenous mechanisms have not been resolved. Here, we analyzed actions of raphé 5-HT neurons on respiratory network activity including at the level of the pre-Bötzinger complex (pre-BötC) in neonatal rat medullary slices in vitro, and in the more intact nervous system of juvenile rats in arterially perfused brainstem-spinal cord preparations in situ. At basal levels of activity, excitation of the respiratory network via simultaneous release of 5-HT and substance P (SP), acting at 5-HT(2A/2C), 5-HT(4), and/or neurokinin-1 receptors, was required to maintain inspiratory motor output in both the neonatal and juvenile systems. The midline raphé obscurus contained spontaneously active 5-HT neurons, some of which projected to the pre-BötC and hypoglossal motoneurons, colocalized 5-HT and SP, and received reciprocal excitatory connections from the pre-BötC. Experimentally augmenting raphé obscurus activity increased motor output by simultaneously exciting pre-BötC and motor neurons. Biophysical analyses in vitro demonstrated that 5-HT and SP modulated background cation conductances in pre-BötC and motor neurons, including a nonselective cation leak current that contributed to the resting potential, which explains the neuronal depolarization that augmented motor output. Furthermore, we found that 5-HT, but not SP, can transform the electrophysiological phenotype of some pre-BötC neurons to intrinsic bursters, providing 5-HT with an additional role in promoting rhythm generation. We conclude that raphé 5-HT neurons excite key circuit components required for generation of respiratory motor output.
The frog skin in vivo is capable of active transepithelial H+ secretion (JH) which is matched by Na+ absorption (JNa). Studies in vitro demonstrate that JH is generated by an H(+)-ATPase pump localized in apical membranes of mitochondria-rich (MR) cells, whereas JNa occurs through an amiloride-sensitive pathway in principal (P) cells. The H+ pump is sensitive to inhibitors of carbonic anhydrase (e.g. acetazolamide) and to specific inhibitors of mitochondrial F1F0 H(+)-ATPase (oligomycin) and vacuolar (V)-type H(+)-ATPase (N-ethylmaleimide) and to inhibitors of both these types of H(+)-ATPases (dicyclohexylcarbodiimide, DCCD). JH is independent of external K+, which differentiates it from gastric H+/K(+)-ATPase and is strictly dependent on aerobic metabolism. The proton pump is primarily implicated in whole-body acid-base regulation. Acute stimulation of JH in response (seconds-minutes) to an acid load involves insertion of H+ pumps (exocytosis) from a cytosolic pool into the apical membrane. The chronic response (days) to metabolic acid load involves morphological changes (increased apical membrane surface area and number of MR cells). Whole-cell patch-clamp recordings of membrane capacitance and current fluctuations from MR cells demonstrate that a respiratory acid load and aldosterone produce rapid exocytotic insertion of DCCD-sensitive conductive membrane. A secondary role of the H+ pump is to energize sodium absorption (JNa) via principal cells from dilute solutions in the absence of a permeant anion under open-circuit conditions. The apparent 1:1 stoichiometry between JH and JNa is a result of transepithelial electrical coupling between these electrogenic fluxes. The H+ pump in MR cells generates a transepithelial current (serosa to apical) which acts as a physiological voltage-clamp to hyperpolarize the apical membrane of P cells. This hyperpolarization can facilitate passive Na+ entry across the apical membrane against a threefold chemical gradient. Since both JH and JNa are sensitive to membrane potential, inhibition or activation of one will produce similar effects on the transport of the other ion. For example, inhibition of JH by ethoxzolamide will reduce JNa. Conversely, blocking JNa with amiloride also inhibits JH. These effects can be avoided or reversed if variations in membrane potential are prevented by voltage-clamping the epithelium. A paradoxical activation of JNa is observed when JH is stimulated by an acid load (CO2), despite inhibition of Na+ channel activity by H+ in P cells.(ABSTRACT TRUNCATED AT 400 WORDS)
We have investigated the neurotransmitters used to control egg-laying in C. elegans. Previous studies suggested that 5-HT released by the HSN motor neurons stimulates egg laying, and that tricyclic antidepressants potentiate egg laying by blocking reuptake of 5-HT by the HSN neurons. We report studies of the wild type and a mutant that lacks detectable 5-HT that suggest 5-HT is not required for egg-laying. Furthermore, we find that ACh is required for egg laying in response to 5-HT, suggesting that 5-HT is not sufficient to activate egg laying. The dominant egl-2(n693) mutation, which causes animals to lay eggs in response to tricyclics but not 5-HT, also conflicts with the model for egg laying. Experiments in which the HSN neurons or 5-HT are removed from egl-2 animals indicate that the action of tricyclics cannot be explained by a block of 5-HT reuptake. We find that D2 family dopamine antagonists can also induce egg laying in egl-2(n693) mutants, and that dopamine inhibits egg laying in the wild type. These results suggest that dominant egl-2 mutations activate an inhibitory dopaminergic pathway that can be blocked by tricyclics and D2 antagonists. We also find that these drugs stimulate egg laying in mutants lacking 5-HT or the HSN neurons, consistent with a target on the egg-laying muscles. In contrast to tricyclics, fluoxetine and other selective 5-HT reuptake inhibitors appear to be specific for 5-HT reuptake in C. elegans egg laying.
Regulating the response of a postsynaptic cell to neurotransmitter is an important mechanism for controlling synaptic strength, a process critical to learning. We have begun to define and characterize genes that may control sensitivity to the neurotransmitter serotonin in the nematode Caenorhabditis elegans by identifying serotonin-hypersensitive mutants. We reported previously that mutations in the gene unc-2, which encodes a putative calcium channel subunit, result in hypersensitivity to serotonin. Here we report that mutants defective in the unc-36 gene, which encodes a homologue of a calcium channel auxiliary subunit, are also serotonin-hypersensitive. Moreover, the unc-36 gene appears to be required in the same cells as unc-2 for control of the same behaviors. Mutations in several other genes, including unc-8, unc-10, unc-20, unc-35, unc-75, unc-77, and snt-1 also result in hypersensitivity to serotonin. Several of these mutations have previously been shown to confer resistance to acetylcholinesterase inhibitors, suggesting that they may affect acetylcholine release. Moreover, we found that mutations that decrease acetylcholine synthesis cause defective egg-laying and serotonin hypersensitivity. Thus, acetylcholine appears to negatively regulate the response to serotonin and may participate in the process of serotonin desensitization.
Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.
A countertransport of H(+) is coupled to Ca(2+) transport across the sarcoplasmic reticulum (SR) membrane. We propose that SR carbonic anhydrase (CA) accelerates the CO(2)-HCO reaction so that H(+) ions, which are exchanged for Ca(2+) ions, are produced or buffered in the SR at sufficient rates. Inhibition of this SR-CA is expected to reduce the rate of H(+) fluxes, which then will retard the kinetics of Ca(2+) transport. Fura 2 signals and isometric force were simultaneously recorded in fiber bundles of the soleus (SOL) and extensor digitorum longus (EDL) from rats in the absence and presence of the lipophilic CA inhibitors L-645151, chlorzolamide (CLZ), and ethoxzolamide (ETZ), as well as the hydrophilic inhibitor acetazolamide (ACTZ). Fura 2 and force signals were analyzed for time to peak (TTP), 50% decay time (t(50)), and their amplitudes. L-645151, CLZ, and ETZ significantly increased TTP of fura 2 by 10-25 ms in SOL and by 5-7 ms in EDL and TTP of force by 6-30 ms in both muscles. L-645151 and ETZ significantly prolonged t(50) of fura 2 and force by 20-55 and 40-160 ms, respectively, in SOL and EDL. L-645151, CLZ, and ETZ also increased peak force of single twitches and amplitudes of fura fluorescence ratio (R(340/380)) at an excitation wavelength of 340 to 380 nm. All effects of CA inhibitors on fura 2 and force signals could be reversed. ACTZ did not affect TTP, t(50), and amplitudes of fura 2 signals or force. L-645151, CLZ, and ETZ had no effects on myosin-, Ca(2+)-, and Na(+)-K(+)-ATPase activities, nor did they affect the amplitude and half-width of action potentials. We conclude that inhibition of SR-CA by impairing H(+) countertransport is responsible for deceleration of intracellular Ca(2+) transients and contraction times.
Area-restricted search (ARS) is a foraging strategy used by many animals to locate resources. The behavior is characterized by a time-dependent reduction in turning frequency after the last resource encounter. This maximizes the time spent in areas in which resources are abundant and extends the search to a larger area when resources become scarce. We demonstrate that dopaminergic and glutamatergic signaling contribute to the neural circuit controlling ARS in the nematode Caenorhabditis elegans. Ablation of dopaminergic neurons eliminated ARS behavior, as did application of the dopamine receptor antagonist raclopride. Furthermore, ARS was affected by mutations in the glutamate receptor subunits GLR-1 and GLR-2 and the EAT-4 glutamate vesicular transporter. Interestingly, preincubation on dopamine restored the behavior in worms with defective dopaminergic signaling, but not in glr-1, glr-2, or eat-4 mutants. This suggests that dopaminergic and glutamatergic signaling function in the same pathway to regulate turn frequency. Both GLR-1 and GLR-2 are expressed in the locomotory control circuit that modulates the direction of locomotion in response to sensory stimuli and the duration of forward movement during foraging. We propose a mechanism for ARS in C. elegans in which dopamine, released in response to food, modulates glutamatergic signaling in the locomotory control circuit, thus resulting in an increased turn frequency.
Small-molecule inhibitors of protein function are powerful tools for biological analysis and can lead to the development of new drugs. However, a major bottleneck in generating useful small-molecule tools is target identification. Here we show that Caenorhabditis elegans can provide a platform for both the discovery of new bioactive compounds and target identification. We screened 14,100 small molecules for bioactivity in wild-type worms and identified 308 compounds that induce a variety of phenotypes. One compound that we named nemadipine-A induces marked defects in morphology and egg-laying. Nemadipine-A resembles a class of widely prescribed anti-hypertension drugs called the 1,4-dihydropyridines (DHPs) that antagonize the alpha1-subunit of L-type calcium channels. Through a genetic suppressor screen, we identified egl-19 as the sole candidate target of nemadipine-A, a conclusion that is supported by several additional lines of evidence. egl-19 encodes the only L-type calcium channel alpha1-subunit in the C. elegans genome. We show that nemadipine-A can also antagonize vertebrate L-type calcium channels, demonstrating that worms and vertebrates share the orthologous protein target. Conversely, FDA-approved DHPs fail to elicit robust phenotypes, making nemadipine-A a unique tool to screen for genetic interactions with this important class of drugs. Finally, we demonstrate the utility of nemadipine-A by using it to reveal redundancy among three calcium channels in the egg-laying circuit. Our study demonstrates that C. elegans enables rapid identification of new small-molecule tools and their targets.
This review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure.
This review capitalizes on the authors' collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models.
Seven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses.
The selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose-response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.
Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been
shown to bind to different carbonic anhydrase isoforms to create a “transport metabolon.” We have expressed NBCe1 derived
from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H+ and Na+ concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes,
the membrane current and the rate of cytosolic Na+ rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2–200 ng). The CAII inhibitor
ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH2-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII
mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H+ change following addition of CO2/ confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity
of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with
NBCe1.
Defects in insulin signaling have been reported in schizophrenia and major depressive disorder, which also share certain negative symptoms such as avolition, anhedonia, and apathy. These symptoms reflect diminished motivational states, which have been modeled in rodents as increased immobility in the forced swimming test. We have discovered that loss-of-function mutations in the insulin receptor (daf-2) and syntaxin (unc-64) genes in Caenorhabditis elegans, brief food deprivation, and exposure to DMSO produce immobility and avolition in non-dauer adults. The animals remain responsive to external stimuli; however, they fail to forage and will remain in place for >12 days or until they die. Their immobility can be prevented with drugs used to treat depression and schizophrenia and that reduce immobility in the forced swimming test. This includes amitriptyline, amoxapine, clozapine, and olanzapine, but not benzodiazepines and haloperidol. Recovery experiments confirm that immobility is induced and maintained by excessive signaling via serotonergic and muscarinic cholinergic pathways. The immobility response described here represents a potential protophenotype for avolition/anhedonia in man. This work may provide clues about why there is a significant increase in depression in patients with diabetes and suggest new therapeutic pathways for disorders featuring diminished motivation as a prominent symptom.
The Na(+) leak-current channel (NALCN) regulates locomotion, respiration and intellectual development. Previous work highlighted striking similarities between characteristic movement phenotypes of NALCN-deficient animals (Drosophila and C. elegans) and the major symptoms of Parkinson's disease and primary progressive freezing gait. We have discovered novel physiological connections between the NALCN, K(+) channels and gap junctions that mediate regulation of locomotion in C. elegans. Drugs that block K(+) channels and gap junctions or that activate Ca(++) channels significantly improve movement of NALCN-deficient animals. Loss of function of the NALCN creates an imbalance in ions, including K(+) and Ca(++) , that interferes with normal cycles of depolarization-repolarization. This work suggests new therapeutic strategies for certain human movement disorders. This article is protected by copyright. All rights reserved.
Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with "DA2A with severe neurological abnormalities" and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with "atypical" forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).
Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Mutations in various genes adversely affect locomotion in model organisms, and thus provide valuable clues about the complex processes that control movement. In C. elegans, loss-of-function mutations in the Na(+) leak current channel (NALCN) and associated proteins (UNC-79 and UNC-80) cause akinesia and fainting (abrupt freezing of movement during escape from touch). It is not known how defects in the NALCN induce these phenotypes or if they are chronic and irreversible. Here, we report that akinesia and freezing are state-dependent and reversible in NALCN-deficient mutants (nca-1;nca-2, unc-79 and unc-80) when additional cation channels substitute for this protein. Two main measures of locomotion were evaluated: spontaneous movement (traversal of > 2 head lengths during a 5 sec observation period) and the touch-freeze response (movement greater than 3 body bends in response to tail touch). Food deprivation for as little as 3 min stimulated spontaneous movement and corrected the touch-freeze response. Conversely, food-deprived animals that moved normally in the absence of bacteria rapidly reverted to uncoordinated movement when re-exposed to food. The effects of food deprivation were mimicked by nicotine, which suggested that acetylcholine mediated the response. Nicotine appeared to act on interneurons or motor neurons rather than directly at the neuromuscular junction because levamisole, which stimulates muscle contraction, did not correct movement. Neural circuits have been proposed to account for the effects of food deprivation and nicotine on spontaneous movement and freezing. The NALCN may play an unrecognized role in human movement disorders characterized by akinesia and freezing gait.
This chapter discusses the interactions between drugs and gene products in Caenorhabditis elegans in genetic pharmacology. Caenorhabditis elegans has been a popular organism for the study of drug action. This chapter discusses the methods that used in compound-based studies of C. elegans and evaluates the effects of compounds on C. elegans growth, development, metabolism, and behavior. The strategies for the isolation, and analysis of drug-resistant and hypersensitive mutants are discussed. Studies combining bioactive compounds and C. elegans can be separated based on experimental strategy. The first strategy employs compounds with known modes of action to characterize particular aspects of C. elegans biology in wild type and mutant animals. The second strategy uses active compounds as screening or selective agents to isolate new drug-resistant or hypersensitive mutants and, thus, to identify genes with altered drug responses. Study of such compound-specific mutants can identify specific drug targets, and/or provide insight into the mechanism of drug action and the sites of drug action. The third strategy involves the use of C. elegans, both wild type and selected mutants, to analyze the mechanism of action of uncharacterized or poorly characterized compounds. This has led to the use of C. elegans as a primary screen for compounds active against parasitic nematodes.
: The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 ± 0.1 nM, a Bmax of 161 ± 27 fmol.mg-1 protein, and a Hill slope of 1.07, at 25°C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: (1) nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; (2) verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50= 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 μM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10-5-10-3M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.
Background:
Infantile neuroaxonal dystrophy (INAD) is a recessive disease that results in total neurological degeneration and death in childhood. PLA2G6 mutation is the underlying genetic defect, but rare genetic heterogeneity has been demonstrated. One of the five families we studied did not link to PLA2G6 locus, and in the family one of the two affected siblings additionally had atypical features including facial dysmorphism, pectus carinatum, scoliosis, pes varus, zygodactyly and bilateral cryptorchidism as well as cerebellar atrophy, as previously reported.
Methods:
Sural biopsy was investigated by electron microscopy. PLA2G6 was screened for mutations by Sanger sequencing. In the mutation-free family, candidate disease loci were found via linkage analysis using data from single nucleotide polymorphism genome scans. Exome sequencing was applied to find the variants at the loci.
Results:
PLA2G6 mutations were identified in four families including the one with an unusually severe phenotype that led to death within the first 2 years of life. In the remaining family, seven candidate loci totalling 15.2 Mb were found and a homozygous truncating mutation p.Q642X was identified in NALCN at 13q32.3. The patients are around 20-years-old.
Conclusions:
NALCN is the gene responsible for INAD with facial dysmorphism. The patients have lived to adulthood despite severe growth and neuromotor retardation. NALCN forms a voltage-independent ion channel with a role in the regulation of neuronal excitability. Our findings broaden the spectrum of genes associated with neuroaxonal dystrophy. Testing infants with idiopathic severe growth retardation and neurodegeneration for NALCN mutations could benefit families.
The molecular modes of action of antipsychotic drugs are poorly understood beyond their effects at the dopamine D2 receptor. Previous studies have placed Akt signaling downstream of D2 dopamine receptors, and recent data have suggested an association between psychotic illnesses and defective Akt signaling. To characterize the effect of antipsychotic drugs on the Akt pathway, we used the model organism C. elegans, a simple system where the Akt/forkhead box O transcription factor (FOXO) pathway has been well characterized. All major classes of antipsychotic drugs increased signaling through the insulin/Akt/FOXO pathway, whereas four other drugs that are known to affect the central nervous system did not. The antipsychotic drugs inhibited dauer formation, dauer recovery, and shortened lifespan, three biological processes affected by Akt signaling. Genetic analysis showed that AKT-1 and the insulin and insulin-like growth factor receptor, DAF-2, were required for the antipsychotic drugs to increase signaling. Serotonin synthesis was partially involved, whereas the mitogen activated protein kinase (MAPK), SEK-1 is a MAP kinase kinase (MAPKK), and calcineurin were not involved. This is the first example of a common but specific molecular effect produced by all presently known antipsychotic drugs in any biological system. Because untreated schizophrenics have been reported to have low levels of Akt signaling, increased Akt signaling might contribute to the therapeutic actions of antipsychotic drugs.
Extracellular K⁺, Na⁺, and Ca²⁺ ions all influence the resting membrane potential of the neuron. However, the mechanisms by which extracellular Na⁺ and Ca²⁺ regulate basal neuronal excitability are not well understood. Recent findings suggest that NALCN, in association with UNC79 and UNC80, contributes a basal Na⁺ leak conductance in neurons. Mutations in Nalcn, Unc79, or Unc80 lead to severe phenotypes that include neonatal lethality and disruption in rhythmic behaviors. This review discusses the properties of the NALCN complex, its regulation, and its contribution to neuronal function and animal behavior.
Whole cell clamp experiments were used to study the mechanism of action of amiloride on cardiac T-type Ca channels in guinea pig ventricular myocytes. Two hundred fifty microM amiloride blocked the T-type Ca channel for approximately 50%, whereas this concentration did not suppress the L-type Ca channel. The Kd value for the block of the T-type Ca channel was 233 microM and the Hill coefficient was 1.1. The drug showed a rapid onset of action and a quick wash out with complete reversibility. Amiloride blocked the T-type Ca channel without frequency-dependency, i.e., block was established at the holding potential (-90 mV) without being dependent on activation and inactivation of the channel. Amiloride reduced the amplitude of the T-type Ca current at all potentials without changing steady-state activation and inactivation. Amiloride did not affect the T-type Ca current when the drug was applied intracellularly via the pipette solution. In summary, amiloride blocked the T-type Ca channel in a channel state- and voltage-independent way.
The binding of [3H]nimodipine to purified synaptic plasma membranes (SPM) isolated from sheep brain cortex was characterized, and the effects of nimodipine, nifedipine, and (+)-verapamil on the [3H]nimodipine binding were compared to the effects on 45Ca2+ translocation under conditions that separate 45Ca2+ fluxes through Ca2+ channels from 45Ca2+ uptake via Na+/Ca2+ exchange. [3H]Nimodipine labels a single class of sites in SPM, with a KD of 0.64 +/- 0.1 nM, a Bmax of 161 +/- 27 fmol X mg-1 protein, and a Hill slope of 1.07, at 25 degrees C. Competition of [3H]nimodipine binding to purified SPM with unlabelled Ca2+ channel blockers shows that: nifedipine and nimodipine are potent competitors, with IC50 values of 4.7 nM and 5.9 nM, respectively; verapamil and (-)-D 600 are partial competitors, with biphasic competition behavior. Thus, (+)-verapamil shows an IC50 of 708 nM for the higher affinity component and the maximal inhibition is 50% of the specific binding, whereas for (-)-verapamil the IC50 is 120 nM, and the maximal inhibition is 30%; (-)-D 600 is even less potent than verapamil in inhibiting [3H]nimodipine binding (IC50 = 430 nM). However, (+)-verapamil, nifedipine, and nimodipine are less potent in inhibiting depolarization-induced 45Ca2+ influx into synaptosomes in the absence of Na+/Ca2+ exchange than in competing for [3H]nimodipine binding. Thus, (+)-verapamil inhibits Ca2+ influx by 50% at about 500 microM, whereas it inhibits 50% of the binding at concentrations 200-fold lower, and the discrepancy is even larger for the dihydropyridines. The Na+/Ca2+ exchange and the ATP-dependent Ca2+ uptake by SPM vesicles are also inhibited by the Ca2+ channel blockers verapamil, nifedipine, and d-cis-diltiazem, with similar IC50 values and in the same concentration range (10(-5)-10(-3) M) at which they inhibit Ca2+ influx through Ca2+ channels. We conclude that high-affinity binding of the Ca2+ blockers by SPM is not correlated with inhibition of the Ca2+ fluxes through channels in synaptosomes under conditions of minimal Na+/Ca2+ exchange. Furthermore, the relatively high concentrations of blockers required to block the channels also inhibit Ca2+ translocation through the Ca2+-ATPase and the Na+/Ca2+ exchanger. In this study, clear differentiation is made of the effects of the Ca2+ channel blockers on these three mechanisms of moving Ca2+ across the synaptosomal membrane, and particular care is taken to separate the contribution of the Na+/Ca2+ exchange from that of the Ca2+ channels under conditions of K+ depolarization.
The nematode Caenorhabditis elegans appears to be a useful model for studying the action of volatile anesthetics. A mutant
strain that is hypersensitive to the widely used anesthetic halothane was described earlier. The mutation is now shown to
be an allele of unc-79. Other alleles of unc-79 are also associated with hypersensitivity to halothane. A strain with a mutation
in a second gene, unc-80, is also hypersensitive to halothane. Nematodes bearing mutations in both unc-79 and unc-80 are slightly
more sensitive to halothane than those bearing only one of these mutations. Mutations in a third gene, unc-9, suppress both
unc-79 and unc-80. Nematodes bearing the suppressor mutations alone have normal sensitivity to halothane. These results show
that sensitivity to halothane can be altered by mutations in several different genes.
The body muscles of the nematode Caenorhabditis elegans contract when the animal is cut in solutions of cholinergic agonists. The pharmacological specificity of the apparent nematode cholinergic receptor is most like a vertebrate nicotink ganglionic receptor. The anthelmintic levamisole resembles nicotine in its effects and acts directly or indirectly as both a cholinergic agonist and antagonist. Mutants at 7 loci conferring extreme resistance to levamisole respond very poorly to cholinergic agonists effective on the wild type. These mutants all share the same uncoordinated motor behavior and contract like the wild type in response to the noncholinergic muscle agonist ouabain. The uncoordinated motor behavior of the mutants and the resistance to levamisole and cholinergic agonists can be copied by exposing the wild type to the cholinergic blocking agent mecamylamine. Another class of mutants (8 loci, 5 corresponding to loci also producing extremely resistant alleles) possesses intermediate resistance to levamisole and cholinergic agonists and behaves pharmacologically and genetically like mutants moderately impaired in the levamisole-sensitive function. A third class of mutants (2 loci) with spasmodic muscle twitching is partially resistant to cholinergic agonists and to ouabain and probably represents defects in the muscle-contraction cycle physiologically downstream from the levamisole-sensitive function. Meta-phenyl-substituted derivatives of levamisole retain considerable biological activity and may be useful in the molecular analysis of our mutants. α-bungarotoxin, benzyltrimethyl-ammonium, and 3-quinuclidinyl benzilate, potential probes of cholinergic receptor function, do not show significant activity in our cut worm assay.
Patients with interstitial deletions of the long arm of chromosome 13 may have widely varying phenotypes. From cytogenetic analysis, we have postulated that there is a discrete region in 13q32 where deletion leads to a syndrome of severe malformations, including digital and brain anomalies. To test this hypothesis at the molecular level, we have studied the deletions in 17 patients; 5 had severe malformations, while the remaining 12 had only minor malformations. Our results indicate that the deletions in the severely affected patients all involve an overlapping region in q32, while the deletions in the mildly affected patients include some, but not all, of this overlapping region. Our findings are consistent with the hypothesis that the severely malformed 13q- phenotype results from the deletion of a critical region in 13q32. This region is presently defined as lying between D13S136 and D13S147 and is on the order of 1 Mb in size.
The class III antiarrhythmic drugs amiodarone and bretylium tosylate are cationic/amphiphilic, and various substances with these physico-chemical properties are known to directly activate heterotrimeric regulatory G proteins. We asked the question of whether class III antiarrhythmic drugs are also direct G protein activators, using HL-60 leukemic cells and purified bovine brain G proteins as model systems. In HL-60 cell membranes, aminodarone increased high affinity GTP hydrolysis with an EC50 of 7.5 microM. The stimulatory effect of amiodarone on GTP hydrolysis was inhibited by pertussis toxin. Amiodarone stimulated binding of guanosine-5'-O-(3-thio)triphosphate to, and incorporation of GTP azidoanilide into, Gi protein alpha subunits in HL-60 membranes. The drug increased the cytosolic Ca2+ concentration in HL-60 cells in the presence but not in the absence of extracellular Ca2+. Amiodarone-induced increases in the cytosolic Ca2+ concentration were reduced by pertussis toxin and by a blocker of non-selective cation channels, SK&F 96365. Amiodarone activated the GTPase of reconstituted Gi/G(o) proteins and G12 with EC50 values of 20 microM and 50 microM, respectively. Bretylium tosylate did not increase GTP hydrolysis in HL-60 membranes or with Gi/G(o) proteins. Our data suggest that amiodarone but not bretylium tosylate is a direct activator of Gi and G(o) proteins and that amiodarone activates nonselective cation channels in HL-60 cells via Gi proteins and independently of Ca2+ mobilization from intracellular stores. Future studies will have to test the hypothesis that direct G protein activation by amiodarone contributes to its toxic and/or therapeutic effects.
The first-generation histamine H1-receptor antagonists, chlorpheniramine (CPHE) and diphenhydramine (DPH), may activate histamine release from basophils and mast cells. Because CPHE and DPH are cationic-amphiphilic and because several substances with such physicochemical properties activate heterotrimeric regulatory guanine nucleotide-binding proteins (G-proteins) in a receptor-independent manner, we asked the question of whether or not H1-receptor antagonists could be G-protein activators as well. In dibutyryl cAMP-differentiated HL-60 cells, CPHE and DPH increased cytosolic Ca2+ concentration and azurophilic granule release in pertussis toxin (PTX)-sensitive manners. In HL-60 membranes, PTX-sensitive stimulations of GTPase [E.C. 3.6.1.] and binding of guanosine 5'-[gamma-thio]triphosphate by H1 receptor antagonists were observed. CPHE and DPH also increased GTP hydrolysis by the purified PTX-sensitive G-protein, transducin. In all-trans-retinoic acid-differentiated HL-60 cells and rat basophilic leukemia cells (RBL 2H3 cells), H1-receptor antagonists induced, unlike in dibutyryl cAMP-differentiated HL-60 cells, Ca2+ influx without Ca2+ mobilization from intracellular stores. CPHE and DPH also induced serotonin release from RBL 2H3 cells. Our data indicate that first-generation H1-receptor antagonists are receptor-independent G-protein activators and that such a mechanism of action accounts for their stimulatory effects in HL-60 cells, basophils, and mast cells.
Unlabelled:
PROPERTIES OF MIBEFRADIL: Mibefradil is a novel calcium channel antagonist with structural and pharmacological characteristics clearly distinct from those of classical calcium antagonists. It is a potent vasodilator with a high selectivity for the coronary vasculature over the peripheral vasculature and the myocardium. Most importantly, this compound can relax vascular muscle and slow the heart rate without reducing cardiac contractility. In addition, it does not stimulate neurohormonal reflexes and it exhibits a good pharmacological profile characterized by a long duration of action.
Mechanism of action:
The mechanism of action of mibefradil is characterized by the selective blockade of transient, low-voltage-activated (T-type) calcium channels over long-lasting, high-voltage-activated (L-type) calcium channels, which is probably responsible for many of its unique properties. CLINICAL USE OF MIBEFRADIL: Although calcium antagonists are mainly used for the treatment of hypertension and angina pectoris, there is strong preclinical evidence that mibefradil may also be beneficial in the treatment of congestive heart failure.
The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is important for synaptic plasticity and nervous system development and function. We have used genetic and electrophysiological methods to demonstrate that NMR-1, a Caenorhabditis elegans NMDA-type ionotropic glutamate receptor subunit, plays a role in the control of movement and foraging behavior. nmr-1 mutants show a lower probability of switching from forward to backward movement and a reduced ability to navigate a complex environment. Electrical recordings from the interneuron AVA show that NMDA-dependent currents are selectively disrupted in nmr-1 mutants. We also show that a slowly desensitizing variant of a non-NMDA receptor can rescue the nmr-1 mutant phenotype. We propose that NMDA receptors in C. elegans provide long-lived currents that modulate the frequency of movement reversals during foraging behavior.
A unique family of putative ion channels that are related to voltage-gated sodium and calcium channels has been identified in genomic and cDNA studies of metazoans. Aside from evidence for expression of family members in the nervous system, little is known about the operation of the channel or its functional significance. In the present study, this conserved family's sole Drosophila member, a gene known both as CG1517 and as Dmalpha1U, is shown to correspond to the narrow abdomen (na) gene and is the locus of a set of mutations that affect sensitivity to anesthetics. Immunohistochemistry of adult heads reveals that the channel is expressed in the neuropil of the central complex and optic lobe; expression is severely depressed in the mutants. In addition to previously described defects, the mutant phenotype is demonstrated here to include dysfunction in the coupling between light and locomotor behavior. Most dramatically, mutant flies have an inversion of relative locomotor activity in light versus dark. The involvement of the channel in daily rhythms of the fruit fly is especially provocative because the human ortholog lies in a candidate region linked to bipolar disorder, a disease frequently associated with altered diurnal behavior.
Caenorhabditis elegans explores its environment by interrupting its forward movement with occasional turns and reversals. Turns and reversals occur at stable frequencies but irregular intervals, producing probabilistic exploratory behaviors. Here we dissect the roles of individual sensory neurons, interneurons, and motor neurons in exploratory behaviors under different conditions. After animals are removed from bacterial food, they initiate a local search behavior consisting of reversals and deep omega-shaped turns triggered by AWC olfactory neurons, ASK gustatory neurons, and AIB interneurons. Over the following 30 min, the animals disperse as reversals and omega turns are suppressed by ASI gustatory neurons and AIY interneurons. Interneurons and motor neurons downstream of AIB and AIY encode specific aspects of reversal and turn frequency, amplitude, and directionality. SMD motor neurons help encode the steep amplitude of omega turns, RIV motor neurons specify the ventral bias of turns that follow a reversal, and SMB motor neurons set the amplitude of sinusoidal movement. Many of these sensory neurons, interneurons, and motor neurons are also implicated in chemotaxis and thermotaxis. Thus, this circuit may represent a common substrate for multiple navigation behaviors.
• chemosensation
• exploratory behavior
• neural circuit
The inhibitory and relaxant effects of the L-type calcium antagonists nifedipine, nimodipine, verapamil and diltiazem, and of the T-type calcium antagonist mibefradil, on contractions of isolated human detrusor muscle were investigated. The tissue was obtained from 10 patients undergoing cystectomy due to bladder cancer. Effects of the calcium antagonists at different concentrations on the concentration-response curves for carbachol were investigated. Furthermore, concentration-relaxation curves were performed using potassium-precontracted muscle strips. All L-type calcium antagonists suppressed the mean concentration-response curve of carbachol significantly at a concentration of 10(-6) M. Mibefradil up to 10(-5) M did not significantly suppress it. Nifedipine significantly reduced the carbachol-induced maximum contraction to 75% and 44%, verapamil to 75% and 67% of the appropriate control value at concentrations of 10(-7) and 10(-6) M, respectively. Diltiazem reduced it insignificantly to 96% and 71% at the above-mentioned concentrations. The concentration-relaxation experiments revealed following pD2-values and maximum relaxations of nifedipine, nimodipine, verapamil and diltiazem, respectively: 6.23, 6.37, 5.66, 5.81 and 85%, 83%, 82%, 90%. Maximum relaxations and pD2-values were not significantly different from each other. The lowest concentration, for which a significant effect compared to control in Student;s t-test was found, amounted to 10(-10) M, 10(-9) M, 10(-7) M, 10(-6.5) M and 10(-4) M for nimodipine, nifedipine, diltiazem, verapamil and mibefradil, respectively. L-type calcium antagonists are very potent relaxant agents of the human detrusor muscle in vitro.
The action of 2-aminoethoxydiphenyl borate (2-APB) on Ca(2+) signalling in HeLa cells and cardiac myocytes was investigated. Consistent with other studies, we found that superfusion of cells with 2-APB rapidly inhibited inositol 1,4,5-trisphosphate (InsP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOC). In addition to abrogating hormone-evoked Ca(2+) responses, 2-APB could antagonise Ca(2+) signals evoked by a membrane permeant InsP(3) ester. 2-APB also slowed the recovery of intracellular Ca(2+) signals consistent with an effect on Ca(2+) ATPases. The inhibitory action of 2-APB on InsP(3) receptors (InsP(3)Rs), SOC channels and Ca(2+) pumps persisted for several minutes after washout of the compound. Application of 2-APB to unstimulated cells had no effect on subsequent Ca(2+) responses suggesting that it has a use-dependent action. Mitochondria in cells treated with 2-APB showed a rapid and slowly reversible swelling. 2-APB did not cause the mitochondria to depolarise, but it reduced the extent of mitochondrial calcium uptake. Although 2-APB has been demonstrated not to affect voltage-operated Ca(2+) channels or ryanodine receptors, we found that it gave a concentration-dependent long-lasting inhibition of Ca(2+) signalling in electrically-stimulated cardiac myocytes, where InsP(3)Rs and SOC channels do not play a significant role. Our data suggest that 2-APB has multiple cellular targets, a use-dependent action, is difficult to reverse and may affect Ca(2+) signalling in cell types where InsP(3) and SOC are not active.
Antipsychotic drugs may produce adverse effects during development in humans and rodents. However, the extent of these effects has not been systematically characterized nor have molecular mechanisms been identified. Consequently, we sought to evaluate the effects of an extensive panel of antipsychotic drugs in a model organism, Caenorhabditis elegans, whose development is well characterized and which offers the possibility of identifying novel molecular targets. For these studies, animals were grown from hatching in the presence of vehicle (control) or antipsychotic drugs over a range of concentrations (20-160microM) and growth was analyzed by measuring head-to-tail length at various intervals. First-generation antipsychotics (e.g., fluphenazine) generally slowed growth and maturation more than second-generation drugs such as quetiapine and olanzapine. This is consistent with in vitro effects on human neuronal cell lines. Clozapine, a second-generation drug, produced similar growth deficits as haloperidol. Converging lines of evidence, including the failure to rescue growth with high concentrations of agonists, suggested that the drug-induced delay in development was not mediated by the major neurotransmitter receptors recognized by the antipsychotic drugs. Moreover, in serotonin-deficient tph-1 mutants, the drugs dramatically slowed development and led to larval arrest (including dauer formation) and neuronal abnormalities. Evaluation of alternative targets of the antipsychotics revealed a potential role for calmodulin and underscored the significance of Ca(2+)-calmodulin signaling in development. These findings suggest that antipsychotic drugs may interfere with normal developmental processes and provide a tool for investigating the key signaling pathways involved.
2-Aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor modulator, inhibits capacitive current transients measured in normal rat kidney and human embryonic kidney 293 cells, an indication of blocking gap junction channels between these cells. Here, we used the dual whole-cell patch-clamp method to study the actions of 2-APB on gap junction channels formed by selected connexins expressed in a communication-deficient neuroblastoma cell line (N2A). 2-APB dose-dependently and reversibly blocked junctional currents of connexin (Cx) 50 gap junction channels. The concentration-inhibition curve of 2-APB on the junctional current indicated an IC(50) of 3.7 microM, lower than that of most gap junction inhibitors. At a concentration of 20 microM, 2-APB also significantly blocked junctional conductance in cell pairs coupled by Cx26, Cx30, Cx36, Cx40, and Cx45 but did not appreciably affect coupling in cell pairs expressing Cx32, Cx43, and Cx46. Although concentration inhibition curves of 2-APB on Cx36 channels were similar to Cx50 (Cx36; IC(50), 3.0 microM), IC(50) values were higher for Cx43 (51.6 microM), Cx45 (18.1 microM), and Cx46 (29.4 microM). The blocking action of 2-APB did not substantially alter transjunctional voltage-dependent gating of Cx50 gap junction channels, and recordings from poorly coupled pairs of Cx50-transfected N2A cells indicated that 2-APB reduced gap junction channel open probability without changing the main state single-channel conductance. The differential efficacy of block by 2-APB of gap junction channels formed by different connexins may provide a useful tool that could be exploited in gap junction research to selectively block certain gap junction channel subtypes.
Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1
• Morgan P.G.
• Sedensky M.
• Meneely P.M.
Multiple sites of action of volatile anesthetics in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 1990; 87: 2965-2969
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, 2
• Campbell D.B.
• Nash H.A.
Use of Drosophila mutants to distinguish among volatile general anesthetics.Proc. Natl. Acad. Sci. USA. 1994; 91: 2135-2139
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]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action.
Serotonin (5-hydroxytryptamine: 5HT) is an important neuroactive substance in the model roundworm, Caenorhabditis elegans. Aside from having effects in feeding and egg-laying, 5HT inhibits motility and also modulates several locomotory behaviors, notably food-induced slowing and foraging. Recent evidence showed that a serotonergic 5HT2-like receptor named SER-1 (also known as 5HT2ce) was responsible for the effect of 5HT on egg-laying. Here we confirm this observation and show that SER-1 also plays an important role in locomotion. A mutant lacking SER-1 was found to be highly resistant to exogenous 5HT in the absence of food and this resistant phenotype was rescued by reintroducing the SER-1 gene in a mutant background. Pharmacological studies showed that the same antagonists that blocked the activity of recombinant SER-1 in vitro also inhibited the effect of 5HT on motility, suggesting the same receptor was responsible for both effects. When tested for locomotory behaviors, the SER-1 mutant was found to be moderately defective in food-induced slowing. In addition, the mutant changed direction more frequently than the wildtype when searching for food, suggesting that SER-1 may play a role in navigational control during foraging. Both these effects required the presence of MOD-1, a 5HT gated chloride channel, and the results indicate that SER-1 and MOD-1 modulate these behaviors through a common pathway. On the basis of expression analysis of a ser-1::GFP translational fusion, SER-1 is prominently located in central, integrating neurons of the head ganglia (RIA and RIC) but not the body wall musculature. The evidence suggests that SER-1 controls locomotion through indirect modulation of neuromuscular circuits and has effects both on speed and direction of movement.
Sodium plays a key role in determining the basal excitability of the nervous systems through the resting "leak" Na(+) permeabilities, but the molecular identities of the TTX- and Cs(+)-resistant Na(+) leak conductance are totally unknown. Here we show that this conductance is formed by the protein NALCN, a substantially uncharacterized member of the sodium/calcium channel family. Unlike any of the other 20 family members, NALCN forms a voltage-independent, nonselective cation channel. NALCN mutant mice have a severely disrupted respiratory rhythm and die within 24 hours of birth. Brain stem-spinal cord recordings reveal reduced neuronal firing. The TTX- and Cs(+)-resistant background Na(+) leak current is absent in the mutant hippocampal neurons. The resting membrane potentials of the mutant neurons are relatively insensitive to changes in extracellular Na(+) concentration. Thus, NALCN, a nonselective cation channel, forms the background Na(+) leak conductance and controls neuronal excitability.