Article

Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

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Abstract

CACNA2D2 is a putative tumor suppressor gene located in the human chromosome 3p21.3 region that shows frequent allelic imbalances in lung, breast, and other cancers. The alpha2delta-2 protein encoded by the gene is a regulatory subunit of voltage-dependent calcium channels and is expressed in brain, heart, and other tissues. Here we report that mice homozygous for targeted disruption of the Cacna2d2 gene exhibit growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells followed by Purkinje cell depletion, enhanced susceptibility to seizures, and cardiac abnormalities. The Cacna2d2(tm1NCIF) null phenotype has much in common with that of Cacna1a mutants, such as cerebellar neuro-degeneration associated with ataxia, seizures, and premature death. A tendency to bradycardia and limited response of null mutants to isoflurane implicate alpha2delta-2 in sympathetic regulation of cardiac function. In summary, our findings provide genetic evidence that the alpha2delta-2 subunit serves in vivo as a component of P/Q-type calcium channels, is indispensable for the central nervous system function, and may be involved in hereditary cerebellar ataxias and epileptic disorders in humans.

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... Purkinje cells (PCs), the primary output pathway from the cerebellar cortex, appear to exclusively and abundantly express the ␣2␦-2 isoform (Cacna2d2) (Barclay et al., 2001;Cole et al., 2005;Lein et al., 2007;Dolphin, 2012), thus providing an opportunity to determine how this protein contributes to synaptic transmission. Like the spontaneous 'ducky' mouse mutants (du 2j /du 2j or du/du), targeted deletion of ␣2␦-2 causes cerebellar ataxia, epilepsy and premature death (Barclay et al., 2001;Brodbeck et al., 2002;Ivanov et al., 2004;Donato et al., 2006), indicative of its importance in neurological function. ...
... We combined structural and electrophysiological analysis of the CF to PC synapse in Cacna2d2 KO mice (Ivanov et al., 2004) to elucidate the contribution of ␣2␦-2 to excitatory synapse formation and transmission. Contrary to positive regulation of excitatory synaptogenesis by the ␣2␦-1 isoform (Li et al., 2004;Eroglu et al., 2009;Chen et al., 2018;Risher et al., 2018), loss of ␣2␦-2 increased functional synaptic innervation by CFs. ...
... Mice were born in the Mendelian ratio of 1:2:1; with WT mice having two copies of the intact Cacna2d2 gene, and KO mice as homozygous mutants. Because ϳ50% of KO mice die prematurely (Ivanov et al., 2004), all experiments used male and female mice between p21-p30, when CF-PC synapses have reached maturity, but before significant loss of KO mice. ...
Article
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α2δ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) predominantly, if not exclusively, express one isoform of this family, α2δ-2 (Cacna2d2), we used PCs as a model system to examine roles of α2δ in excitatory synaptic function in male and female Cacna2d2 knockout mice. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked excitatory postsynaptic currents (EPSCs). CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2+ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wildtype as a result of increased glutamate re-uptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of α2δ proteins.Significance Statementα2δ proteins (Cacna2d1-4) regulate synaptic transmission and synaptogenesis, but co-expression of multiple α2δ isoforms has obscured a clear understanding of how various α2δ proteins control synaptic function. We focused on roles of the α2δ-2 protein (Cacna2d2), whose deletion causes cerebellar ataxia and epilepsy in mice and humans. Because cerebellar Purkinje cells only expresses this single isoform, we studied excitatory climbing fiber synaptic function onto Purkinje cells in Cacna2d2 knockout mice. Using optical and electrophysiological analysis, we provide a detailed description of the changes in Purkinje cells lacking α2δ-2, and provide a comprehensive mechanistic explanation for how functional synaptic phenotypes contribute to the altered cerebellar output.
... Previously, mice lacking the CACNA2D2 gene, a putative TSP receptor, were found to be extremely sensitive to PTZ. Absence of CACNA2D2 causes epilepsy in mice and a severe form of epilepsy in children (Ivanov et al., 2004;Edvardson et al., 2013;Pippucci et al., 2013). We performed qPCR test to measure expression levels of CACNA2D1 and CACNA2D2 in the brains of TSP1, TSP2 and TSP1/2 KO and WT mice (Figure 4). ...
... The α2δ subunits help direct and stabilize the voltage gated calcium channels to the presynaptic region and drive synaptic vesicle release (Hoppa et al., 2012). CACNA2D1 is expressed ubiquitously through different tissues and has robust expression in neuronal tissues and excitatory cells; CACNA2D2 is highly expressed in GABAergic interneurons (Ellis et al., 1988;Gong et al., 2001;Cole at al., 2005).Transgenic deletion of CACNA2D2 causes seizures and increases PTZ seizure sensitivity (Barclay et al., 2001;Brodbeck et al., 2002;Brill et al., 2004;Ivanov et al., 2004). TSP1 deletion in our study caused a decrease in CACNA2D2 mRNA, α2δ−1/2 protein, and increased seizure sensitivity to PTZ though no direct causal relationship could be proven. ...
... In contrast, mutating the gabapentin α 2 δ−2 receptor binding site had minimal impact on pain or seizure therapy, suggesting that suppressing α 2 δ−2 is not the therapeutic mechanism of gabapentin. Several mouse models with loss of CACNA2D2 result in epilepsy phenotypes and several families have been reported with CACNA2D2 mutations and severe epilepsy (Barclay et al., 2001;Brodbeck et al., 2002;Brill et al., 2004;Ivanov et al., 2004;Edvardson et al., 2013;Pippucci et al., 2013). Mechanistically inhibiting α 2 δ−1 and increasing α 2 δ−2 activity might be a better mechanism for treating epilepsy. ...
Article
The development of a hyperexcitable neuronal network is thought to be a critical event in epilepsy. Thrombospondins (TSPs) regulate synaptogenesis by binding the neuronal α2δ subunit of the voltage-gated calcium channel. TSPs regulate synapse formation during development and in the mature brain following injury. It is unclear if TSPs are involved in hyperexcitability that contributes to the development of epilepsy. Here we explore the development of epilepsy using a pentylenetetrazole (PTZ) kindling model in mice lacking TSP1 and TSP2. Unexpectedly, we found increased sensitivity to PTZ kindling in mice lacking TSP1, while mice lacking TSP2 kindled similar to wild-type. We found that the increased seizure susceptibility in the TSP1 knockout (KO) mice was not due to a compensatory increase in TSP2 mRNA as TSP1/2 KO mice were sensitive to PTZ, similar to the TSP1 KO mice. Furthermore, there were similar levels of TGF-B signal activation during kindling in the TSP1 KO mice compared to wild-type. We observed decreased expression of voltage-dependent calcium channel subunit CACNA2D1 mRNA in TSP1, TSP2, and TSP1/2 KO mice. Decreased CACNA2D2 mRNA was only detected in mice that lacked TSP1 and α2δ-1/2 protein levels in the cortex was lower in the TSP 1/2 KO mice. CACNA2D2 knockout mice have spontaneous seizures and increased PTZ seizure susceptibility. Here we report similar findings, TSP1, and TSP1/2 KO mice have low levels of CACNA2D2 mRNA expression and α2δ-1/2 receptor level in the cortex, and are more susceptible to seizures. CACNA2D2 mutations in mice and humans can cause epilepsy. Our data suggests TSP1 in particular may control CACNA2D2 levels and could be a modifier of seizure susceptibility. Copyright © 2015. Published by Elsevier Ltd.
... Using the hypothalamic A11 dopaminergic circuit as a platform, we explain the potential therapeutic actions of dopaminergics, opioids, adenosine analogs, and calcium channel ␣ 2 ␦ ligands ( fig. 2 ). It is important to note that the hypotha- [112,113,115,117,123,125]. ␣ 2 ␦ subunits: [116,142,144,145,147,[150][151][152]. GBP = Gabapentin; IML = intermediolateral nucleus; PGB = pregabalin. ...
... Unlike dopamine-, opioid-, and adenosine-receptor ligands, ␣ 2 ␦ ligands do not require G-protein-coupled, cAMP-mediated second messenger pathways to exert their effects on voltage-gated calcium channels. ␣ 2 ␦ -containing calcium channels are found at various locations within the proposed RLS circuit ( fig. 2 ), including the dorsal horn [144] , dorsal root ganglia neurons [139,142,[144][145][146][147][148][149] , intermediolateral neurons [150] , and sympathetic pre-and postganglionic neurons [151,152] . Thus, they are in a prime position to control the excitability of the circuitry at the various points that may be involved in the symptomatology of RLS. ...
... Thus, they are in a prime position to control the excitability of the circuitry at the various points that may be involved in the symptomatology of RLS. Sympathetic spinal ganglia also express ␣ 2 ␦ subunits [152] . Furthermore, expression of presynaptic ␣ 2 ␦ subunits is induced by injury [146] and ␣ 2 ␦ ligands inhibit voltage-gated channel activity in dor-sal root ganglia neurons [138] and reduce sympathetically maintained pain at the spinal level [151] . ...
... When heterologously expressed, all α 2 δ subunit isoforms can modulate the trafficking and/or the current properties of Ca V α 1 subunits (reviewed in . Accordingly all α 2 δ subunits have been shown to be involved in Ca 2+ channel-dependent functions in skeletal and cardiac muscle (α 2 δ-1; Tuluc et al., 2007;Gach et al., 2008;, in the cerebellum (α 2 δ-2, Ivanov et al., 2004;Donato et al., 2006;Senatore et al., 2012), and in other CNS synapses (α 2 δ-1; Hoppa et al., 2012; α 2 δ-3; ; α 2 δ-4; Wycisk et al., 2006). ...
... This was also suggested by previous correlation analyses of VGCC mRNA abundance . However, unlike the situation in the cerebellum, where Ca V 2.1 α 1 subunits (mediating P/Q currents) and α 2 δ-2 are the dominating isoforms (Ivanov et al., 2004;Donato et al., 2006;Senatore et al., 2012), overall α 2 δ-2 expression is much lower in hippocampus Fig. 4F). α 2 δ-1 and α 2 δ-3 knock-out mice display only mild if any overall CNS phenotypes . ...
... α 2 δ-1 and α 2 δ-3 knock-out mice display only mild if any overall CNS phenotypes . In contrast, α 2 δ-2 knockout or mutant (ducky) mice display epilepsy and ataxia and show severely impaired cerebellar development Ivanov et al., 2004). Thus, our findings on the differential synaptic roles of α 2 δ isoforms are in agreement with the phenotypes of isoform specific α 2 δ subunit null mice. ...
Thesis
Full-text available
Auxiliary α2δ subunits regulate the current density and the kinetics of voltage-gated calcium channels (VGCCs). Of the four α2δ isoforms known, three (α2δ-1, -2, -3) are expressed in the central nervous system. For example, α2δ-2 mutants suffer from severe neurological phenotypes like ataxia, epilepsy, and impaired synapse formation. Here we addressed the question as to whether α2δs contribute to synaptic release and if so, which α2δ isoform preferentially interacts with presynaptic P/Q- and N-type calcium channels. Synaptic release properties were determined by the loss of FM dye fluorescence during sustained depolarization of cultured hippocampal neurons. To identify which VGCC subtype is predominant in synaptic release, the contributions of presynaptic calcium channel isoforms were dissected. Application of the P/Q-type channel blocker ω-agatoxin IVA and the N-type channel blocker ω-conotoxin GVIA revealed that presynaptic P/Q-type channels dominated the FM dye release properties during sustained depolarization of cultured hippocampal neurons were dominated by presynaptic P/Q-type channels although N-type channels were present in 85 % of the synapses. Release in neurons derived from α2δ-3 knockout mice and treated with a pharmacological blocker of α2δ-1/-2 (gabapentin, GBP) was severely reduced compared to untreated α2δ-3 deficient neurons. α2δ-3 deficiency aggravated the effect of GBP, implying that the observed effect of GBP was mediated by block of α2δ-1 and -2.Expression of α2δ-3 partially restored release. Surprisingly, siRNA mediated knockdown of the most abundant α2δ isoform in cultured hippocampal neurons, α2δ-1, had no effect on the release. Vice versa, overexpression of α2δ-1 reduced the extent of synaptic release. As α2δ-3 compensated for blocked α2δ-1 and -2 in the GBP experiments, α2δ-1 knockdown was repeated in neurons derived from α2δ-3 deficient mice. Release was equally efficient in α2δ-1 and -3 double deficient neurons compared to α2δ-3 knockout neurons. N-type channel dependent release was slightly enhanced in α2δ-1 and -3 double deficient neurons, indicating that the remaining α2δ-2 subunit replaced less efficient α2δ isoforms. As synaptic release was impaired when α2δ-2 was blocked by GBP but not by α2δ-1 shRNA alone nor by α2δ-1/-3 double deficiency, evidence builds up suggestive of α2δ-2 as the major presynaptic α2δ isoform. As functional synapses are a prerequisite for the study of synaptic release, Tthe role of α2δ subunits in synaptogenesis were was revealed investigated by quantification of co-localization of the immunolabeled presynaptic marker synapsin with the postsynaptic marker PSD95. Synapse formation was modified but remained functional close to normal in α2δ-1 shRNA treated α2δ-3 knockout neurons. In contrast, gabapentinChronic GBP treatment left synapse formationsynaptogenesis unaffected. Together our results suggest that α2δ-2 is an essential constituent of presynaptic calcium channel complexes. As P/Q-type channels predominately regulated synaptic release, we conclude that and therefore is a α2δ-2 is the preferential interaction partner of P/Q-type channels.
... These CS were in keeping with generalized tonic-clonic seizures described for rodent models in the past (Velíšková 2006). Previous studies have reported similar CS in other mouse models (Ivanov et al., 2004;Muramatsu et al., 2008;Silva-Fernandes et al., 2010;El-Hayek et al., 2011a). Subtle or ambiguous convulsive behaviors, such as head nodding, jerking, and/or shaking were not classified as CS. ...
... Behaviorally, the CS we observed from both aging and adult mice resemble the generalized tonic-clonic seizures described in previous rodent models (Velíšková 2006;Ivanov et al., 2004;Muramatsu et al., 2008;Silva-Fernandes et al., 2010;El-Hayek et al., 2011a). ...
Article
Stroke is the leading cause of seizures and epilepsy in the aged population, with post-stroke seizures being a poor prognostic factor. The pathological processes underlying post-stroke seizures are not well understood and studies of these seizures in aging/aged animals remain scarce. Therefore, our primary objective was to model post-stroke seizures in aging mice (C57 black strain, 16-20 month-old), with a focus on early-onset, convulsive seizures that occur within 24-hours of brain ischemia. We utilized a middle cerebral artery occlusion model and examined seizure activity and brain injury using combined behavioral and electroencephalographic monitoring and histological assessments. Aging mice exhibited vigorous convulsive seizures within hours of the middle cerebral artery occlusion. These seizures manifested with jumping, rapid running, barrel-rolling and/or falling all in the absence of hippocampal-cortical electrographic discharges. Seizure development was closely associated with severe brain injury and acute mortality. Anticonvulsive treatments after seizure occurrence offered temporary seizure control but failed to improve animal survival. A separate cohort of adult mice (6-8 months-old) exhibited analogous early-onset convulsive seizures following the middle cerebral artery occlusion but had better survival outcomes following anticonvulsive treatment. Collectively, our data suggest that early-onset convulsive seizures are a result of severe brain ischemia in aging animals. Copyright © 2015. Published by Elsevier Inc.
... Existing mouse models with spontaneous mutations and targeted deletions for all individual α 2 δ subunits enabled novel insights into their potential functional redundancy as well as specificity: deletion of α 2 δ-1 impaired synaptic NMDA receptor (NMDAR) recruitment, excitatory synaptogenesis, and spine morphology (Risher et al., 2018), and LTP-associated learning and memory (Zhou et al., 2018). Loss of full-length α 2 δ-2 in four distinct mouse strains with naturally occurring mutations [ducky: Barclay et al., 2001;Brodbeck et al., 2002;entla: Brill et al., 2004;and ducky(2J): Donato et al., 2006] and targeted deletions in Cacna2d2 (Ivanov et al., 2004) induced epilepsy, dyskinesia, cerebellar atrophy, and high mortality in juvenile mice. α 2 δ-3 knockout mice display altered pain processing (Neely et al., 2010), abnormal hearing (Pirone et al., 2014), anxiety-like behavior (Landmann et al., 2018a), and sensory cross-activation (Neely et al., 2010;Landmann et al., 2018b). ...
... These reports indicate that α 2 δ-1 either exerts specific functions or that low amounts of the remaining isoforms likely do not suffice to compensate for the loss of α 2 δ-1. Along those lines, the severe consequences of α 2 δ-2 mutations in animal models (Barclay et al., 2001;Brill et al., 2004;Ivanov et al., 2004;Donato et al., 2006) are likely caused by its predominant expression in the cerebellum (Schlick et al., 2010). Notably, α 2 δ-3 knockout mice display overexpression of Ca V 2 calcium channel subtypes in the brain, possibly resulting in enhanced neuronal excitability (Landmann et al., 2018b). ...
Article
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Auxiliary α2δ subunits of voltage-gated calcium channels modulate channel trafficking, current properties, and synapse formation. Three of the four isoforms (α2δ-1, α2δ-2, and α2δ-3) are abundantly expressed in the brain; however, of the available knockout models, only α2δ-2 knockout or mutant mice display an obvious abnormal neurological phenotype. Thus, we hypothesize that the neuronal α2δ isoforms may have partially specific as well as redundant functions. To address this, we generated three distinct α2δ double knockout mouse models by crossbreeding single knockout (α2δ-1 and -3) or mutant (α2δ-2/ducky) mice. Here, we provide a first phenotypic description and brain structure analysis. We found that genotypic distribution of neonatal litters in distinct α2δ-1/-2, α2δ-1/-3, and α2δ-2/-3 breeding combinations did not conform to Mendel’s law, suggesting premature lethality of single and double knockout mice. Notably, high occurrences of infant mortality correlated with the absence of specific α2δ isoforms (α2Δ-2 > α2δ-1 > α2δ-3), and was particularly observed in cages with behaviorally abnormal parenting animals of α2δ-2/-3 cross-breedings. Juvenile α2δ-1/-2 and α2δ-2/-3 double knockout mice displayed a waddling gate similar to ducky mice. However, in contrast to ducky and α2δ-1/-3 double knockout animals, α2δ-1/-2 and α2δ-2/-3 double knockout mice showed a more severe disease progression and highly impaired development. The observed phenotypes within the individual mouse lines may be linked to differences in the volume of specific brain regions. Reduced cortical volume in ducky mice, for example, was associated with a progressively decreased space between neurons, suggesting a reduction of total synaptic connections. Taken together, our findings show that α2δ subunits differentially regulate premature survival, postnatal growth, brain development, and behavior, suggesting specific neuronal functions in health and disease.
... We extended those findings here to show that heterozygous Puralpha (+/−) mice display gait ataxias, such as wider-based steps, less overlap in forepaws and hindpaws, and increased foot drags and missteps, as well as more outwardly rotated hindpaws during forward walking. Such gait changes are typically associated with cerebellar degeneration (Ivanov et al., 2004, Takeuchi et al., 2012, matching our findings of neuronal loss in the cerebellar vermis of heterozygous Pur-alpha (+/−) mice. These mice also showed limb and abdomen hypotonia during SHIRPA testing, changes also typically attributed to the cerebellum. ...
... These mice also showed limb and abdomen hypotonia during SHIRPA testing, changes also typically attributed to the cerebellum. The increased foot drags and outward hindpaw rotation during forward walking could also be related to peripheral nerve degeneration (Sereda et al., 1996), the possibility of which we are exploring in a separate study, although similar gait changes have been observed in association with cerebellar dysfunction (Ivanov et al., 2004, Takeuchi et al., 2012. Protein-altering de novo mutations in PURA, which encodes the Pur-alpha protein, have recently been described in several young human subjects (Hunt et al., 2014, Lalani et al., 2014, Tanaka et al., 2015. ...
Article
Pur-alpha is a highly conserved sequence specific DNA and RNA binding protein with established roles in DNA replication, RNA translation, cell cycle regulation, and maintenance of neuronal differentiation. Prior studies have shown that mice lacking Pur-alpha (-/-) display decreased neurogenesis and impaired neuronal differentiation. We sought to examine for the first time, the behavioral phenotype and brain histopathology of mice that are heterozygous (+/-) for Pur-alpha. Standardized behavioral phenotyping revealed a decreased escape response to touch, limb and abdominal hypotonia, and gait abnormalities in heterozygous Pur-alpha (+/-) mice, compared to wild-type (+/+) littermates. Footprint pattern analyses showed wider-based steps, increased missteps and more outwardly rotated hindpaws in heterozygous Pur-alpha (+/-) mice, suggestive of cerebellar pathology. Barnes maze and novel object location testing revealed significant memory deficits in heterozygous Pur-alpha mice, suggestive of hippocampal pathology. Quantitative immunohistochemical assays of the vermal region of the cerebellum and CA1-3 regions of the hippocampus revealed reduced numbers of neurons in general, as well as reduced numbers of Pur-alpha+-immunopositive neurons and dendrites in heterozygous Pur-alpha mice, compared to wild type littermates. Past studies have implicated mutations in Pur-alpha in several diseases of brain development and neurodegeneration. When combined with these new findings, the Pur-alpha heterozygous knockout mice may provide an animal model in which to study mechanisms of and treatments for Pur-alpha related cognitive deficiencies and neuropathology.
... Interestingly, this study provided evidence that the defect in synapse formation was independent from the Drosophila pore forming α 1 subunit (cacophony), because in contrast to the stj mutants, cacophony null mutants did not show a defect in synapse formation. α 2 δ-2 mutant (ducky, ducky 2J ) and knockout mice display altered Purkinje cell morphology and reduced calcium currents (Barclay et al. 2001;Ivanov et al. 2004;Donato et al. 2006), which suggests a role in maintaining normal cellular physiology and likely also the morphology of synapses. A spontaneous mouse mutation of α 2 δ-4 (Cacna2d4) causes structural and functional abnormalities of retinal ribbon synapses associated with the loss of rods (Wycisk et al. 2006a). ...
... Taken together the role of α 2 δ-1 for CNS disease remains unclear and the resulting phenotypes and severities may be associated with alterations in the overall neuronal excitability, which can be influenced by a variety of other factors. α 2 δ-2: There are several strains of naturally occurring mice with mutations in the Cacna2d2 gene (ducky, ducky 2j , and entla) (Barclay et al. 2001;Brodbeck et al. 2002;Brill et al. 2004;Donato et al. 2006) as well as a targeted α 2 δ-2 knockout mouse (Ivanov et al. 2004), which result in a loss of the full-length α 2 δ-2 protein (ducky, ducky 2j , knockout) or a structurally altered protein (entla). Affected homozygous mice show a decreased life span, infertility, and reduced body size when compared to heterozygous or wildtype littermates. ...
Article
In nerve cells the ubiquitous second messenger calcium regulates a variety of vitally important functions including neurotransmitter release, gene regulation, and neuronal plasticity. The entry of calcium into cells is tightly regulated by voltage-gated calcium channels, which consist of a heteromultimeric complex of a pore forming α1, and the auxiliary β and α2δ subunits. Four genes (Cacna2d1-4) encode for the extracellular membrane-attached α2δ subunits (α2δ-1 to α2δ-4), out of which three isoforms (α2δ-1 to -3) are strongly expressed in the central nervous system. Over the years a wealth of studies has demonstrated the classical role of α2δ subunits in channel trafficking and calcium current modulation. Recent studies in specialized neuronal cell systems propose roles of α2δ subunits beyond the classical view and implicate α2δ subunits as important regulators of synapse formation. These findings are supported by the identification of novel human disease mutations associated with α2δ subunits and by the fact that α2δ subunits are the target of the anti-epileptic and anti-allodynic drugs gabapentin and pregabalin. Here we review the recently emerging evidence for specific as well as redundant neuronal roles of α2δ subunits and discuss the mechanisms for establishing and maintaining specificity.
... Na + /K + -ATPase (encoded by the Atp1b2 gene) maintain resting potential and effective transport, and regulate cellular volume (50). Furthermore, CACNα2δ channels (encoded by the Cacna2d2, Cacna2d3 and Cacng8 genes) are present in the membrane of excitable cells, including that of cardiac muscle (51). Cardiac RYR2 (encoded by the Ryr2 gene) mediates the sarcoplasmic release of stored calcium ions during cardiac calcium-induced calcium release (52). ...
Article
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Suxiao Jiuxin Pill (SX), a traditional Chinese medicine compound consisting primarily of tetramethylpyrazine and borneol, has been reported to protect against ischemic heart disease. However, the effects of SX on mitochondrial injury and gene expression in various signaling pathways are unclear. The aim of the present study was to investigate the effects of SX on mitochondrial injury and to screen the expression of genes potentially altered by SX using a cell culture model of ischemic injury. Simulated ischemia was established by culturing HL‑1 cardiomyocytes in Dulbecco’s modified Eagle’s medium without glucose or serum in a hypoxic chamber containing 95% N2and 5% CO2for 24 h. HL‑1 cardiomyocytes were divided into 3 groups: Control, ischemic injury and ischemic injury + SX (100 μg/ml; n=3 wells/group). Mitochondrial membrane potential was detected by staining with JC‑1 dye. The mRNA expression levels of adenylyl cyclase (Adcy) 1‑9, adrenoceptor β1, Akt1, ATPase Na+/K+ transporting subunit β2, calcium voltage‑gated channel auxiliary subunit α2δ (Cacna2d)2, Cacna2d3, calcium channel voltage‑dependent γ subunit 8, cytochrome C oxidase subunit 6A2 (Cox6a2), fibroblast growth factor receptor (Fgfr) 4, Fgf8, Fgf12, Gnas complex locus, glycogen synthase kinase 3β (Gsk3b), mitogen‑activated protein kinase (Mapk)11‑14, Mapk kinase kinase kinase 1 (Map4k1), Mas1, nitric oxide synthase 3 (Nos3), phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit α (Pik3ca), phospholipase A2 group 4A, rap guanine nucleotide exchange factor 4 and ryanodine receptor 2 were detected using reverse transcription‑quantitative polymerase chain reaction. The protein expression levels of phosphoinositide 3‑kinase (PI3K), MAS‑1 and phosphorylated‑endothelial NOS were also examined by immunofluorescence staining. The decrease in mitochondrial membrane potential in the cell culture model of ischemic injury (P<0.001) was significantly attenuated by SX treatment (P<0.001). Furthermore, increases in the mRNA expression levels of Adcy2 (P<0.05), 3 (P<0.01) and 8 (P<0.05) in the ischemic injury model were significantly attenuated by SX treatment (P<0.01), and SX treatment significantly decreased the mRNA expression levels of Adcy1 (P<0.01) and 6 (P<0.05) in ischemic cells. Decreases in the mRNA expression levels of Cox6a2 (P<0.001), Gsk3b (P<0.01) and Pik3ca (P<0.001) in the ischemic injury model were also significantly attenuated by SX treatment (P<0.05, P<0.01 and P<0.001, respectively). In addition, the decrease in the protein expression of PI3K (P<0.001) was significantly attenuated by SX treatment (P<0.001). The present findings indicate that SX may protect cardiomyocytes against mitochondrial injury and attenuate alterations in the gene expression of Adcy2, 3 and 8, Cox6a2, Gsk3b and Pik3ca during ischemic injury.
... These findings indicate that α2δ-1 subunit has an important role in neuropathic pain states and the analgesic effects of gabapentinoids are mediated through the α2δ-1 subunit. In contrast, α2δ-2 subunit is dominantly expressed in the cerebellar Purkinje cells, and mutant mice with a α2δ-2 subunit deletion have been reported to show ataxia, paroxysmal dyskinesia and absence seizures (Barclay et al., 2001;Brodbeck et al., 2002;Brill et al., 2004;Ivanov et al., 2004;Donato et al., 2006). Furthermore, human epileptic encephalopathies associated with α2δ-2 mutations have been reported (Edvardson et al., 2013;Pippucci et al., 2013). ...
Article
Mirogabalin ([(1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl]acetic acid), a novel ligand for the α2δ subunit of voltage-gated calcium channels, is being developed to treat pain associated with diabetic peripheral neuropathy and postherpetic neuralgia. In the present study, we investigated the in vitro binding characteristics and in vivo analgesic effects of mirogabalin, compared to those of pregabalin, a standard α2δ ligand. Mirogabalin showed potent and selective binding affinities for the α2δ subunits, while having no effects on 186 off-target proteins. Similar to pregabalin, mirogabalin did not show clear subtype selectivity (α2δ-1 vs α2δ-2) or species differences (human vs rat). However, in contrast to pregabalin, mirogabalin showed greater binding affinities for human α2δ-1, human α2δ-2, rat α2δ-1, and rat α2δ-2 subunits; further, it had a slower dissociation rate for the α2δ-1 subunit than the α2δ-2 subunit. Additionally, in experimental neuropathic pain models, partial sciatic nerve ligation rats and streptozotocin-induced diabetic rats, mirogabalin showed more potent and longer lasting analgesic effects. In safety pharmacological evaluations, mirogabalin and pregabalin inhibited rota-rod performance and locomotor activity in rats; however, the safety indices of mirogabalin were superior to those of pregabalin. In conclusion, mirogabalin shows potent and selective binding affinities for the human and rat α2δ subunits, and slower dissociation rates for the α2δ-1 subunit than the α2δ-2 subunit. It shows potent and long-lasting analgesic effects in rat models of neuropathic pain, and wider safety margins for side effects of the central nervous system. These properties of mirogabalin can be associated with its unique binding characteristics.
... This is a sign of neurological abnormality, cooccurring with seizures or increased seizure susceptibility in several mouse models for neurological diseases. 30,31 To test whether abnormal electrical activity is present in brains of MLC mice, we performed wireless ECoG recordings with an electrode placed above the motor cortex in both MLC mouse models (Fig 3A). Long-term recordings (48 hours) from mice in their home cage revealed that both MLC lines show a significantly higher occurrence of abnormal discharges than wild-type mice (Fig 3B-E). ...
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Objective: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking. Methods: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole cell patch-clamp recordings and K+-sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC. Results: An early onset of seizures is common in MLC. Similarly, in MLC mice we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+dynamics and neuronal network activity are abnormal in MLC mice. Interpretation: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. This article is protected by copyright. All rights reserved.
... The safety margins of mirogabalin were approximately 2.4-5.3 fold higher than those of pregabalin. Several reports have shown that the α 2 δ-1 subunit is associated with induction and maintenance of NeP, while the α 2 δ-2 subunit is related to ataxia, dyskinesia, and absence seizures, respectively [40,[53][54][55][56]. The higher safety margin of mirogabalin could be related to the slower dissociation rate of mirogabalin for the α 2 δ-1 subunit than the α 2 δ-2 subunit compared with those of pregabalin, as described in subsection 5.1.1. ...
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Introduction Neuropathic pain (NeP) is a chronic and refractory condition in many patients, and its treatment is a challenge for physicians. A new voltage-gated Ca²⁺ channel α2δ ligand, mirogabalin, has a high specific binding affinity for the α2δ subunit, with a slower dissociation rate for α2δ-1 than α2δ-2 compared to that of pregabalin. Mirogabalin was shown to be effective in NeP animal models, with a margin of safety between central nervous system side effects and the analgesic effect of the dose. It exerted a favorable analgesic effect, was well tolerated in patients with peripheral NeP (P-NeP), and was first approved in Japan in 2019 and subsequently in Korea and Taiwan in 2020. Areas covered The purpose of this article is to review the pharmacological characteristics, pharmacokinetics, and efficacy and safety of mirogabalin for NeP based on the results of non-clinical and clinical studies. Expert opinion Although there are several first-line therapies for NeP, insufficient efficacy and adverse drug reactions of NeP drugs often cause patient dissatisfaction. Mirogabalin was effective and well tolerated with a step-wise dose increase in clinical studies on P-NeP patients. Thus, mirogabalin is expected to be a useful treatment option for patients with P-NeP.
... Histopathological observation based on H&E staining and IHC showed the destruction of cerebellum granule layer and astrocytes activation in AgNPs-treated rat cerebellum, which provided histopathological evidences on the neurotoxicity of AgNPs. Similar findings were reported that the cerebellum granule cell density was reduced in cerebellar ataxia mice with Ca 2+ P/Q channelopathies (Ivanov et al., 2004). Voltage-dependent ion channels play important roles in generating the rhythmic membrane potential behavior of neuron and regulating a diversity of cellular processes. ...
... Additionally, all of the reported patients experience multiple treatment-resistant seizure types without focality and involuntary movements. Of note is the consistent observation of cerebellar atrophy (Figure 1(a)), which is also reported in several mouse models of Cacna2d2 dysfunction [6,7]. Moreover, it is interesting to note the occurrence of prolonged seizures and photosensitivity for both our patient and the patient described by Pippucci and colleagues [3]. ...
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CACNA2D2 encodes an auxiliary subunit of the voltage-dependent calcium channel. To date, there have only been two reports of individuals with early-infantile epileptic encephalopathy due to CACNA2D2 mutations. In both reports, patients were homozygous for the identified variants. Here, we report a patient with epileptic encephalopathy and cerebellar atrophy who was found to have two novel variants in the CACNA2D2 gene: c.782C>T (p.Pro261Leu) and c.3137T>C (p.Leu1046Pro), by whole-exome sequencing. The variants were shown to be inherited in trans and the unaffected parents were confirmed to be heterozygous carriers. This is the third report of recessive CACNA2D2 variants associated with disease and the first report of compound heterozygous variants. The clinical description of this new case highlights the phenotypic similarities amongst individuals with CACNA2D2 -related disease and suggests that CACNA2D2 should be considered as a differential diagnosis in individuals with cerebellar dysfunction and multiple seizure types that begin in the first year of life.
... Another spontaneous mutation of the Cacna2d2 gene (entla) encodes a mutant form of the α 2 δ-2 protein with an intact C-terminus. This mutation also causes an atypical form of generalized absence epilepsy in mice [51], as does a targeted knockout of Cacna2d2 [52]. The epileptic phenotype of mice lacking α 2 δ-2 is counterintuitive if one hypothesizes that the α 2 δ subunit positively modulates T-type calcium channels in neurons. ...
Article
Background Spike-wave discharges, underlying absence seizures, are generated within a cortico-thalamo-cortical network that involves the somatosensory cortex, the reticular thalamic nucleus, and the ventrobasal thalamic nuclei. Activation of T-type voltage-sensitive calcium channels (VSCCs) contributes to the pathological oscillatory activity of this network, and some of the first-line drugs used in the treatment of absence epilepsy inhibit T-type calcium channels. The α2δ subunit is a component of high voltage-activated VSCCs (i.e., L-, N-, P/Q-, and R channels) and studies carried out in heterologous expression systems suggest that it may also associate with T channels. The α2δ subunit is also targeted by thrombospondins, which regulate synaptogenesis in the central nervous system. Objective To discuss the potential role for the thrombospondin/α2δ axis in the pathophysiology of absence epilepsy. Methods We searched PubMed articles for the terms “absence epilepsy”, “T-type voltage-sensitive calcium channels”, “α2δ subunit”, “ducky mice”, “pregabalin”, “gabapentin”, “thrombospondins”, and included papers focusing this Review's scope. Results We moved from the evidence that mice lacking the α2δ-2 subunit show absence seizures and α2δ ligands (gabapentin and pregabalin) are detrimental in the treatment of absence epilepsy. This suggests that α2δ may be protective against absence epilepsy via a mechanism that does not involve T channels. We discuss the interaction between thrombospondins and α2δ and its potential relevance in the regulation of excitatory synaptic formation in the cortico-thalamo-cortical network. Conclusion We speculate on the possibility that the thrombospondin/α2δ axis is critical for the correct functioning of the cortico-thalamo-cortical network, and that abnormalities in this axis may play a role in the pathophysiology of absence epilepsy.
... Another mutant mouse, entla, with a similar epileptic phenotype, was then identified and found to have a duplication of exon 3 in cacna2d2 (Brill et al. 2004). Mice with a targeted gene deletion in cacna2d2 also show an epileptic and ataxic phenotype (Ivanov et al. 2004). The mutation in ducky and ducky 2J mice is associated with abnormal morphology of the Purkinje cells (Brodbeck et al. 2002) and markedly attenuated spontaneous activity in these neurons (Donato et al. 2006). ...
Article
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Voltage-gated calcium channels are essential players in many physiological processes in excitable cells. There are three main subdivisions of calcium channel, defined by the pore-forming α1 subunit, the CaV 1, CaV 2 and CaV 3 channels. For all the subtypes of voltage-gated calcium channel, their gating properties are key for the precise control of neurotransmitter release, muscle contraction and cell excitability, among many other processes. For the CaV 1 and CaV 2 channels, their ability to reach their required destinations in the cell membrane, their activation and the fine tuning of their biophysical properties are all dramatically influenced by the auxiliary subunits that associate with them. Furthermore, there are many diseases, both genetic and acquired, involving voltage-gated calcium channels. This review will provide a general introduction and then concentrate particularly on the role of auxiliary α2 δ subunits in both physiological and pathological processes involving calcium channels, and as a therapeutic target. This article is protected by copyright. All rights reserved.
... These findings indicate that the a 2 d-1 subunit has an important role in neuropathic pain states and that the analgesic effects of gabapentinoids are mediated through the a 2 d-1 subunit. In contrast, the a 2 d-2 subunit is dominantly expressed in the cerebellar Purkinje cells, and mutant mice with a a 2 d-2 subunit deletion have been reported to show ataxia, paroxysmal dyskinesia, and absence seizures (Barclay et al., 2001;Brodbeck et al., 2002;Brill et al., 2004;Ivanov et al., 2004;Donato et al., 2006). Furthermore, human epileptic encephalopathies associated with a 2 d-2 mutations have been reported (Edvardson et al., 2013;Pippucci et al., 2013). ...
... This is a sign of neurological abnormality, cooccurring with seizures or increased seizure susceptibility in several mouse models for neurological diseases. 30,31 To test whether abnormal electrical activity is present in brains of MLC mice, we performed wireless ECoG recordings with an electrode placed above the motor cortex in both MLC mouse models (Fig 3A). Long-term recordings (48 hours) from mice in their home cage revealed that both MLC lines show a significantly higher occurrence of abnormal discharges than wild-type mice (Fig 3B-E). ...
Article
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OBJECTIVE: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood-onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking. METHODS: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole-cell patch-clamp recordings and K+-sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC. RESULTS: An early onset of seizures is common in MLC. Similarly, in MLC mice, we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+dynamics and neuronal network activity are abnormal in MLC mice. INTERPRETATION: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. Ann Neurol 2018;83:636-649.
... A subunidade δ é formada por um único segmento transmembrana junto ao domínio IV da subunidade 1c (De Jongh et al., 1989). A expressão incorreta destas subunidades pode levar ao desenvolvimento de bradicardias (Ivanov et al., 2004). Quando as subunidades 2 e δ são expressas em conjunto com a subunidade 1c ocorre um incremento dos sítios de ligação do Receptor Diidropiridínico (DHPR) e das correntes iônicas (Singer et al., 1991;Wei et al., 1995;Bangalore et al., 1996). ...
Thesis
The eugenol, is an essential oil extracted from cloves , and is widely used in dentistry and in the food industry . Literature data show that in intact papillary, the eugenol at high concentrations (5 mM), induces the increase in resting tension . The same can be observed in preparations of permeabilized skeletal muscle cells, and such contractions, partially inhibited by 2-APB, a blocker of IP3R. In this work we evaluated the ability of eugenol to increase the resting tension of atrial trabeculae muscle is independent of Ca2+. Wistar rats (250 -300g) rats were used and anesthetized by inhalation of diethyl ether. After thoracotomy, the hearts were removed and perfused with Ringer's solution for proper dissection. After being dissected, the trabeculae were immersed in 2.5 ml tubs (Ringer 281°C) aerated (oxygen ) pH 7.4. In the technique of intact trabeculae, the atria were stimulated by rectangular pulses (0.5Hz). In the technique of permeabilized fiber (skinned fibers) trabeculae were dissected Solution R (Table 1) then permeabilized by saponin and/or Triton X-100. In intact trabeculae a concentration-response curve to eugenol was carried out in normal Ringer solution. The experiments with intact atrial trabeculae were repeated using channel blockers releasing Ca2+ from the SR in the absence and presence of eugenol in normal ringer's and free-Ca2+ ringer solution. In the presence of Procaine and Ryanodine, blocking the release of Ca2+ from the SR, both in normal ringer's and free-Ca2+ ringer solution, eugenol was able to induce an increase in resting tension. A total inhibition of the ability of eugenol increase in resting tension was possible with the use of BDM, an inhibitor of cyclization of contractile proteins. After the exhaustion of the stock of Ca2+ from SR by caffeine in freeCa2+ ringer solution, eugenol was able to increase the resting tension. The data suggest that the will acts by which eugenol is independent of Ca2+ stored in the SR, but is the result of an active state cyclization of cross-bridges. In permeabilized trabeculae, the ruthenium red was not able to inhibit the increase in resting tension. Just as in intact trabeculae BDM was able to reverse the resting tension induced eugenol. Even in the presence of high concentration of EGTA eugenol is able to induce an increase in resting tension. When the trabeculae were permeabilized by Triton X-100 resting tension is reversed, it was observed that the trabeculae were permeabilized by saponin for 80 minutes. Treatment with colchicine potentiate the effect of eugenol. The results of experiments with arsenite suggest that one of the ways in which eugenol is acting via inhibition of p38 MAPK and the ATP measurement experiments indicate that another possible way of action of eugenol is by reducing this compound in solution. The results together indicate that the activity of eugenol is not directly on the contractile proteins, acts by a Ca2+ independent pathway, appears to act by a cellular pathway that may be associated with microtubules. As it seems, eugenol, does not act through a single wire, second our results, it can act through the p38 MAPK pathway and via reduction of free ATP. Keywords: eugenol , atrial trabeculae , force in the absence of calcium
... A subunidade δ é formada por um único segmento transmembrana junto ao motifs IV da subunidade  1c (De Jongh et al., 1989). A expressão incorreta destas subunidades pode levar ao desenvolvimento de bradicardias (Ivanov et al., 2004). Quando as subunidades  2 e δ são expressas em conjunto com a subunidade  1c ocorre um incremento dos sítios de ligação do Receptor Diidropiridínico (DHPR) e das correntes iônicas (Singer et al., 1991;Wei et al., 1995;Bangalore et al., 1996). ...
... Entla (ent) mice result from an in-frame duplication in Cacna2d2 and resemble the phenotype of ducky mice except that entla mice do not show neuroanatomical or central nervous system abnormalities. 25 Ligand-binding assays using gabapentin on cerebellar Purkinje cells isolated from entla mice showed >60% reduced binding to the membrane compared to wildtype and heterozygous mice demonstrating that a2d-2 contributes to gabapentin binding. ...
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Objective: To characterize the molecular and clinical phenotypic basis of developmental and epileptic encephalopathies caused by rare biallelic variants in CACNA2D2. Methods: Two affected individuals from a family with clinical features of early onset epileptic encephalopathy were recruited for exome sequencing at the Centers for Mendelian Genomics to identify their molecular diagnosis. GeneMatcher facilitated identification of a second family with a shared candidate disease gene identified through clinical gene panel-based testing. Results: Rare biallelic CACNA2D2 variants have been previously reported in three families with developmental and epileptic encephalopathy, and one family with congenital ataxia. We identified three individuals in two unrelated families with novel homozygous rare variants in CACNA2D2 with clinical features of developmental and epileptic encephalopathy and cerebellar atrophy. Family 1 includes two affected siblings with a likely damaging homozygous rare missense variant c.1778G>C; p.(Arg593Pro) in CACNA2D2. Family 2 includes a proband with a homozygous rare nonsense variant c.485_486del; p.(Tyr162Ter) in CACNA2D2. We compared clinical and molecular findings from all nine individuals reported to date and note that cerebellar atrophy is shared among all. Interpretation: Our study supports the candidacy of CACNA2D2 as a disease gene associated with a phenotypic spectrum of neurological disease that include features of developmental and epileptic encephalopathy, ataxia, and cerebellar atrophy. Age at presentation may affect apparent penetrance of neurogenetic trait manifestations and of a particular clinical neurological endophenotype, for example, seizures or ataxia.
... In cortical tissue, some studies suggest that Ca V α 2 δ-2 is more abundant in interneurons than in glutamatergic neurons [274]. Deleterious effects from disrupting Ca V α 2 δ-2 have been observed in mouse genetic models including ataxia and seizures, however none of them are directly related to psychiatric disorders [8,[281][282][283][284][285]. Purcell et al., found three de novo variations in CACNA2D2 in patients with SCZ. ...
Article
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Psychiatric disorders are mental, behavioral or emotional disorders. These conditions are prevalent, one in four adults suffer from any type of psychiatric disorders world-wide. It has always been observed that psychiatric disorders have a genetic component, however, new methods to sequence full genomes of large cohorts have identified with high precision genetic risk loci for these conditions. Psychiatric disorders include, but are not limited to, bipolar disorder, schizophrenia, autism spectrum disorder, anxiety disorders, major depressive disorder, and attention-deficit and hyperactivity disorder. Several risk loci for psychiatric disorders fall within genes that encode for voltage-gated calcium channels (CaVs). Calcium entering through CaVs is crucial for multiple neuronal processes. In this review, we will summarize recent findings that link CaVs and their auxiliary subunits to psychiatric disorders. First, we will provide a general overview of CaVs structure, classification, function, expression and pharmacology. Next, we will summarize tools to study risk loci associated with psychiatric disorders. We will examine functional studies of risk variations in CaV genes when available. Finally, we will review pharmacological evidence of the use of CaV modulators to treat psychiatric disorders. Our review will be of interest for those studying pathophysiological aspects of CaVs.
... GBP is a member of the antiepileptic [11,12], anxiolytic [13] and antinociceptive [14,15] drugs of the gabapentinoid family, including GBP, pregabalin and mirogabalin [4,[16][17][18][19][20][21][22][23]. While the pharmacological activity of GBP is mediated by α 2 δ subunits, it is also prevented by site-directed mutations [24] in either α 2 δ1 (1103 aa, GenBank accession number: O08532) or α 2 δ2 (1156 aa, GenBank accession number: NP_001167518) that are able to abolish GBP binding, although α 2 δ3 does not bind GBP [3,10]. ...
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For voltage-gated Ca2+ channel (VGCC), its α2δ subunits are traditionally considered to be auxiliary subunits that regulates VGCC trafficking to the plasma membrane. The antiepileptic, antinociceptive and anxiolytic gabapentin (GBP) has previously been shown to bind the VGCC α2δ subunits with high affinity to disrupt VGCC trafficking. Yet, the interaction between GBP and α2δ still remains poorly understood from a structural point of view. For instance, it is not clear yet what the structural implication is of α2δ-1-bound GBP against VGCC trafficking. With a set of experimental data-driven structural analysis of the VGCC α2δ-1 and its ligand GBP, this article postulates for the first time that: 1), α2δ-1 bound GBP stabilizes the α2δ-1-GBP complex structure; 2), α2δ-1 bound GBP restrains the conformational flexibility of α2δ-1; 3), α2δ-1-bound GBP establishes an electrostatic axis consisting of Q535 (Gln535)-R241 (Arg241)-GBP (gabapentin)-D452 (Asp452), which constitutes an energetically favourable contribution towards the structural stability of the α2δ-1-GBP complex and helps restrains the conformational flexibility and local structural rigidification of α2δ-1; and 4), GBP-induced local conformational inflexibility and structural rigidification of α2δ-1 is one key step in the pharmacological disruption of VGCC trafficking by GBP.
... AKT2 is a critical regulator for cardiomyocyte survival and metabolism [33]. CACNA2D2 plays an important role in heart rate [34], and the CACNA2D2-knockout mouse showed lower heart rate [35]. TCF4 contributes to muscle fiber and basement membrane recovery following the muscle fiber damage induced by exercise [36]. ...
Article
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Background: Fuzhong buffalo, a native breed of Guangxi Zhuang Autonomous Region, is traditionally used as a draft animal to provide farm power in the rice cultivation. In addition, the Fuzhong buffalo also prepared for the bullfighting festival organized by the locals. The detection of the selective signatures in its genome can help in elucidating the selection mechanisms in its stamina and muscle development of a draft animal. Results: In this study, we analyzed 27 whole genomes of buffalo (including 15 Fuzhong buffalo genomes and 12 published buffalo genomes from Upper Yangtze region). The ZHp, ZFst, π-Ratio, and XP-EHH statistics were used to identify the candidate signatures of positive selection in Fuzhong buffalo. Our results detected a set of candidate genes involving in the pathways and GO terms associated with the response to exercise (e.g., ALDOA, STAT3, AKT2, EIF4E2, CACNA2D2, TCF4, CDH2), immunity (e.g., PTPN22, NKX2-3, PIK3R1, ITK, TMEM173), nervous system (e.g., PTPN21, ROBO1, HOMER1, MAGI2, SLC1A3, NRG3, SNAP47, CTNNA2, ADGRL3). In addition, we also identified several genes related to production and growth traits (e.g., PHLPP1, PRKN, MACF1, UCN3, RALGAPA1, PHKB, PKD1L). Our results depicted several pathways, GO terms, and candidate genes to be associated with response to exercise, immunity, nervous system, and growth traits. Conclusions: The selective sweep analysis of the Fuzhong buffalo demonstrated positive selection pressure on potential target genes involved in behavior, immunity, and growth traits, etc. Our findings provided a valuable resource for future research on buffalo breeding and an insight into the mechanisms of artificial selection.
... It is unclear if the impaired electrical activity associated with reduced α 2 δ-2 expression in SH-SY5Y overexpressing CLN1 relates to the abnormal firing of neuronal cells in other experimental settings. It is worth remarking, however, that biallelic variants in CACNA2D2 are detected in an early onset form of epileptic encephalopathy, which shares some clinical features with CLN1 disease (Brill et al., 2004;Ivanov et al., 2004;Punetha et al., 2019). Recent evidence suggests that the effects of CACNA2D2 on axonal growth and elongation as well as on synaptic formation are inhibited by pregabalin, a drug which targets the α 2 δ-2 subunit and which is used in several neurological conditions, including epilepsy (Tedeschi et al., 2016). ...
Article
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CLN1 disease (OMIM #256730) is an inherited neurological disorder of early childhood with epileptic seizures and premature death. It is associated with mutations in CLN1 coding for Palmitoyl-Protein Thioesterase 1 (PPT1), a lysosomal enzyme which affects the recycling and degradation of lipid-modified (S-acylated) proteins by removing palmitate residues. Transcriptomic evidence from a neuronal-like cellular model derived from differentiated SH-SY5Y cells disclosed the potential negative roles of CLN1 overexpression, affecting the elongation of neuronal processes and the expression of selected proteins of the synaptic region. Bioinformatic inquiries of transcriptomic data pinpointed a dysregulated expression of several genes coding for proteins related to voltage-gated ion channels, including subunits of calcium and potassium channels (VGCC and VGKC). In SH-SY5Y cells overexpressing CLN1 (SH-CLN1 cells), the resting potential and the membrane conductance in the range of voltages close to the resting potential were not affected. However, patch-clamp recordings indicated a reduction of Ba2+ currents through VGCC of SH-CLN1 cells; Ca2+ imaging revealed reduced Ca2+ influx in the same cellular setting. The results of the biochemical and morphological investigations of CACNA2D2/α2δ-2, an accessory subunit of VGCC, were in accordance with the downregulation of the corresponding gene and consistent with the hypothesis that a lower number of functional channels may reach the plasma membrane. The combined use of 4-AP and NS-1643, two drugs with opposing effects on Kv11 and Kv12 subfamilies of VGKC coded by the KCNH gene family, provides evidence for reduced functional Kv12 channels in SH-CLN1 cells, consistent with transcriptomic data indicating the downregulation of KCNH4. The lack of compelling evidence supporting the palmitoylation of many ion channels subunits investigated in this study stimulates inquiries about the role of PPT1 in the trafficking of channels to the plasma membrane. Altogether, these results indicate a reduction of functional voltage-gated ion channels in response to CLN1/PPT1 overexpression in differentiated SH-SY5Y cells and provide new insights into the altered neuronal excitability which may underlie the severe epileptic phenotype of CLN1 disease. It remains to be shown if remodeling of such functional channels on plasma membrane can occur as a downstream effect of CLN1 disease.
... It is unclear if the impaired electrical activity associated with reduced α 2 δ-2 expression in SH-SY5Y overexpressing CLN1 relates to the abnormal firing of neuronal cells in other experimental settings. It is worth remarking, however, that biallelic variants in CACNA2D2 are detected in an early onset form of epileptic encephalopathy, which shares some clinical features with CLN1 disease (Brill et al., 2004;Ivanov et al., 2004;Punetha et al., 2019). Recent evidence suggests that the effects of CACNA2D2 on axonal growth and elongation as well as on synaptic formation are inhibited by pregabalin, a drug which targets the α 2 δ-2 subunit and which is used in several neurological conditions, including epilepsy (Tedeschi et al., 2016). ...
Article
Full-text available
CLN1 disease (OMIM # 256730 ) is an inherited neurological disorder of early childhood with epileptic seizures and premature death. It is associated with mutations in CLN1 coding for Palmitoyl-Protein Thioesterase 1 (PPT1), a lysosomal enzyme which affects the recycling and degradation of lipid-modified (S-acylated) proteins by removing palmitate residues. Transcriptomic evidence from a neuronal-like cellular model derived from differentiated SH-SY5Y cells disclosed the potential negative roles of CLN1 overexpression, affecting the elongation of neuronal processes and the expression of selected proteins of the synaptic region. Bioinformatic inquiries of transcriptomic data pinpointed a dysregulated expression of several genes coding for proteins related to voltage-gated ion channels, including subunits of calcium and potassium channels (VGCC and VGKC). In SH-SY5Y cells overexpressing CLN1 (SH- CLN1 cells), the resting potential and the membrane conductance in the range of voltages close to the resting potential were not affected. However, patch-clamp recordings indicated a reduction of Ba ²⁺ currents through VGCC of SH- CLN1 cells; Ca ²⁺ imaging revealed reduced Ca ²⁺ influx in the same cellular setting. The results of the biochemical and morphological investigations of CACNA2D2/α 2 δ-2, an accessory subunit of VGCC, were in accordance with the downregulation of the corresponding gene and consistent with the hypothesis that a lower number of functional channels may reach the plasma membrane. The combined use of 4-AP and NS-1643, two drugs with opposing effects on K v 11 and K v 12 subfamilies of VGKC coded by the KCNH gene family, provides evidence for reduced functional K v 12 channels in SH- CLN1 cells, consistent with transcriptomic data indicating the downregulation of KCNH4 . The lack of compelling evidence supporting the palmitoylation of many ion channels subunits investigated in this study stimulates inquiries about the role of PPT1 in the trafficking of channels to the plasma membrane. Altogether, these results indicate a reduction of functional voltage-gated ion channels in response to CLN1 /PPT1 overexpression in differentiated SH-SY5Y cells and provide new insights into the altered neuronal excitability which may underlie the severe epileptic phenotype of CLN1 disease. It remains to be shown if remodeling of such functional channels on plasma membrane can occur as a downstream effect of CLN1 disease.
... This applied mainly to the α2δ-1 subunit of VGCCs: the dissociation half-life from the α2δ-1 subunit is 11.1 h, and from the α2δ-2 subunit, it is 2.4 h for mirogabalin vs. 1.4 h from both α2δ-1 and α2δ-2 subunits for pregabalin [19]. These differences explain the diverse efficacy and safety profiles of mirogabalin compared to other gabapentinoids because the α2δ-1 subunits of VGCCs have been linked with gabapentinoids analgesic properties [9], whereas the α2δ-2 subunits of VGCCs with the adverse CNS effects [22]. According to experimental data, mirogabalin's safety profile is superior to pregabalin's, which is evidenced by the comparison of dosages that produce 50% of the maximum adverse effect and 50% of maximum analgesia, respectively [19]. ...
Article
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The efficacy of neuropathic pain control remains unsatisfactory. Despite the availability of a variety of therapies, a significant proportion of patients suffer from poorly controlled pain of this kind. Consequently, new drugs and treatments are still being sought to remedy the situation. One such new drug is mirogabalin, a selective ligand for the α2δ subunits of voltage-gated calcium channels (VGCC) developed by Sankyo group for the management of neuropathic pain. In 2019 in Japan, mirogabalin was approved for peripheral neuropathic pain following the encouraging results of clinical trials conducted with diabetic peripheral neuropathic pain (DPNP) and postherpetic neuralgia (PHN) patients. The ligand selectivity of mirogabalin for α2δ-1 and α2δ-2 and its slower dissociation rate for α2δ-1 than for α2δ-2 subunits of VGCC may contribute to its strong analgesic effects, wide safety margin, and relatively lower incidence of adverse effects compared to pregabalin and gabapentin. This article discusses the mechanism of action of mirogabalin, presents data on its pharmacodynamics and pharmacokinetics, and reviews the available experimental and clinical studies that have assessed the efficacy and safety of the drug in the treatment of selected neuropathic pain syndromes.
... Loss-of-function mutations in three separate genes that contribute to the normal function of this heteromeric channel have been identified. These include multiple mutant alleles of (1) the pore-forming a 1 membrane subunit (CACNA1A), (2) the alpha subunit interaction domain of the cytoplasmic b4 subunit (CACNB4) which binds to the a 1 subunit and specifically regulates the activity of P/Q-type calcium channels (Burgess et al., 1997;Walker et al., 1999), and (3) the a 2 d subunit (CACNA2D2) with transmembrane and extracellular domains which anchor the channel to the membrane and interact directly with the a 1 subunit of the P/Qtype calcium channel (Ivanov et al., 2004). These mutations lead to a loss of calcium 233 6 P/Q-type calcium channels: selective impairment of inhibitory release? ...
Article
Absence epilepsy is a common disorder that arises in childhood and can be refractory to medical treatment. Single genetic mutations in mice, at times found in patients with absence epilepsy, provide the unique opportunity to bridge the gap between dysfunction at the genetic level and pathological oscillations within the thalamocortical circuit. Interestingly, unlike other forms of epilepsy, only genes related to ion channels have so far been linked to absence phenotypes. Here, we delineate a paradigm which attempts to unify the various monogenic models based on decades of research. While reviewing the particular impact of these individual mutations, we posit a framework involving fast feedforward disinhibition as one common mechanism that can lead to increased tonic inhibition in the cortex and/or thalamus. Enhanced tonic inhibition hyperpolarizes principal cells, deinactivates T-type calcium channels, and leads to reciprocal burst firing within the thalamocortical loop. We also review data from pharmacologic and polygenic models in light of this paradigm. Ultimately, many questions remain unanswered regarding the pathogenesis of absence epilepsy.
... Mouse model of prostate cancer [151] Gabapentin is a ligand of Cav1.3 [151] Apoptotic pathway [278] [281]. COS-7 cells for spinocerebellar ataxia type 2 [164]. ...
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... Entla is another mouse strain with a mutation in cacna2d2, and these mice display ataxia and tonic-clonic epilepsy (Brill et al., 2004). This is also seen in the targeted knockout of cacna2d2 (Ivanov et al., 2004). These mutations are all recessive, with the heterozygotes showing no significant behavioral effects (Barclay et al., 2001). ...
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Andrew J Thorpe, PhD, is a Director in the Department of CNS and Inflammation Chemistry, Pfizer Global Research and Development, Ann Arbor, MI, USA. He obtained his BSc in Medicinal Chemistry from University College London in 1990 and a PhD in organic synthesis from the University of Exeter in 1993, working under the guidance of Prof Stanley M Roberts, where his focus was on the synthesis of novel nucleoside analogs. From Exeter he moved to a Postdoctoral Research Associate position with Prof Tomas Hudlicky at Virginia Polytechnic Institute and State University and later at the University of Florida, where he studied the chemistry of cyclohexadiene-cis-diols. After his research in the Hudlicky group, he moved to Eli Lilly and Company where he served as a Postdoctoral Research Fellow under the mentorship of Tony Shuker in Endocrine Research. He then joined Parke-Davis Research (now Pfizer Global Research and Development) in Ann Arbor, Michigan in the CNS department where he studied the chemistry and pharmacology of α2–δ ligands. His research experience is extended beyond CNS and incorporates that of inflammatory diseases.
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Genes for familial hemiplegic migraine (FHM) and episodic ataxia type-2 (EA-2) have been mapped to chromosome 19p13. We characterized a brain-specific P/Q-type Ca2+ channel α1-subunit gene, CACNL1A4, covering 300 kb with 47 exons. Sequencing of all exons and their surroundings revealed polymorphic variations, including a (CA)n-repeat (D19S1150), a (CAG)n-repeat in the 3′-UTR, and different types of deleterious mutations in FHM and EA-2. In FHM, we found four different missense mutations in conserved functional domains. One mutation has occurred on two different haplotypes in unrelated FHM families. In EA-2, we found two mutations disrupting the reading frame. Thus, FHM and EA-2 can be considered as allelic channelopathies. A similar etiology may be involved in common types of migraine.
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Voltage-dependent Ca2+ channels are modulated by complex interactions with the α2δ subunit. In vitro translation was used to demonstrate a single transmembrane topology of the α2δ subunit in which all but the transmembrane sequence and 5 carboxy-terminal amino acids are extracellular. The glycosylated extracellular domain is required for current stimulation, as shown by coexpression of truncated α2δ subunits with α1A and β4 subunits in Xenopus oocytes and deglycosylation with peptide-N-glycosidase F. However, coexpression of the transmembrane domain-containing δ subunit reduced the stimulatory effects of full-length α2δ subunits and substitution of a different transmembrane domain resulted in a loss of current stimulation. These results support a model whereby the α2δ transmembrane domain mediates subunit interactions and the glycosylated extracellular domain enhances current amplitude.
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Ducky, a recessive autosomal mutation in mice, causes growth retardation, incoordination, excitability, seizures, and shortened lifespan. Neuropathological examination revealed dysgenesis of selective central nervous system regions. The most severely underdeveloped and deficient parts were the spinal cord, spinal ganglia and spinal nerves, the cerebellum and medulla oblongata. Among related structures the pons and trapezoid body, and dorsal root nuclei were greatly reduced in size, the degree of severity following this order. Also, there was Purkinje cell disease, characterized by deaths and losses, and axonal dystrophy and myelin deficiency were found in several fiber tracts, i.e., lateral lemniscus, and spinocerebellar, vestibulospinal and cerebellospinal tracts. Based on spino-medullo-cerebellar dysgenesis and consisting mainly of spinal and pontocerebellar hypoplasia combined with Purkinje cell atrophy, the symptoms in ducky could best be described collectively as a locomotor ataxia or dyskinesia. The seizure susceptibility was not explained. In view of the clinico-pathological findings the ducky syndrome might be analogous to forms of human spinocerebellar degenerations.
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The molecular underpinnings of cerebellar development are being established through the identification of naturally occurring mutated genes and the knockout of other genes. Sets of genes expressed in the regions of the mes- and metencephalon have been shown to play a crucial role in specifying the cerebellar anlage. Other genes have been shown to be crucial to early granule-cell development, migration of Purkinje and granule cells, and neuron–glia interactions. However, the process of development will ultimately be understood in terms of cellular interactions and the roles that each cell type plays in the assembly of cerebellar structure. One of the most important interactions is between granule and Purkinje cells. This relationship has been shown to be crucial for the control of cell number, migration of neuroblasts and cell Differentiation.
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Mice with the mutant gene tottering (tg, chromosome 8, autosomal recessive) show, in adolescence, abnormal bursts of bilaterally synchronous spike waves as revealed in electrocorticograms recorded over long periods. The spike waves are accompanied by behavioral "absence" attacks and intermittent focal motor seizures showing somatotopic progression. Cerebral metabolic activity during seizures was assayed by autoradiography of brain sections from mice injected intravenously with 14C-labeled 2-deoxyglucose. Metabolic activity was increased bilaterally in selected brainstem structures. Spontaneous electrocorticographic and clinical seizures of this general pattern were recognized hitherto only in humans.
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Measurement of the ability of mice to balance on a rotating rod or cone is often used as a measure of impaired motor function. In most of these procedures the mice must be trained prior to the test. In the new screen test described in this paper, untrained mice are used in a 60 sec test which measures the ability of mice to either climb to the top of or cling to the bottom of a horizontal screen. The ED50 values obtained for failure to reach the top of the horizontal screen are similar to those obtained with the rotarod; the values for falling from the screen are somewhat higher. With both of the horizontal screen measures there were fewer control failures than in the rotarod procedure.
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Hemodynamic and electrophysiologic studies were performed in 30 survivors of sudden cardiac arrest with hypertrophic cardiomyopathy (HC) to determine responsible factors. Electrophysiologic abnormalities alone were present in 27 patients (90%): sinus node dysfunction in 14 (47%), delayed atrio-ventricular nodal conduction in 1 (3%), abnormal His-Purkinje conduction in 7 (23%), an inducible atrial tachycardia in 7 (23%), and inducible sustained ventricular arrhythmia in 21 (70%). Sustained ventricular arrhythmia was polymorphic ventricular tachycardia (VT) in 18 patients (86%), monomorphic VT in 2 patients (7%) and ventricular fibrillation in 1 patient (3%). In 1 patient the arrhythmia recorded during an episode of cardiac arrest and induced at electrophysiologic study was polymorphic VT. VT was induced with less than or equal to 2 extra-stimuli in only 1 patient (3%) but with less than or equal to 3 extra-stimuli in 20 patients (97%). Potential causes of sudden cardiac arrest were found in all patients and were multiple in 13 patients (43%). These were (1) ventricular electrical instability in 21 patients (70%), (2) severe left ventricular outflow tract obstruction in 8 patients (27%), (3) bradycardia in 5 patients (17%), (4) myocardial ischemia associated with hypotension in 5 patients (17%), and (5) atrial tachycardia resulting in hypotension in 4 patients (13%). Of the 21 patients with inducible sustained ventricular arrhythmia, 17 received an implantable defibrillator device and 4 were treated with antiarrhythmic drugs. Seven patients underwent left ventricular septal myectomy.(ABSTRACT TRUNCATED AT 250 WORDS)
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We have presented recommendations for the optimum acquisition of quantitative two-dimensional data in the current echocardiographic environment. It is likely that advances in imaging may enhance or supplement these approaches. For example, three-dimensional reconstruction methods may greatly augment the accuracy of volume determination if they become more efficient. The development of three-dimensional methods will depend in turn on vastly improved transthoracic resolution similar to that now obtainable by transesophageal echocardiography. Better resolution will also make the use of more direct methods of measuring myocardial mass practical. For example, if the epicardium were well resolved in the long-axis apical views, the myocardial shell volume could be measured directly by the biplane method of discs rather than extrapolating myocardial thickness from a single short-axis view. At present, it is our opinion that current technology justifies the clinical use of the quantitative two-dimensional methods described in this article. When technically feasible, and if resources permit, we recommend the routine reporting of left ventricular ejection fraction, diastolic volume, mass, and wall motion score.
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To determine the accuracy of echocardiographic left ventricular (LV) dimension and mass measurements for detection and quantification of LV hypertrophy, results of blindly read antemortem echocardiograms were compared with LV mass measurements made at necropsy in 55 patients. LV mass was calculated using M-mode LV measurements by Penn and American Society of Echocardiography (ASE) conventions and cube function and volume correction formulas in 52 patients. Penn-cube LV mass correlated closely with necropsy LV mass (r = 0.92, p less than 0.001) and overestimated it by only 6%; sensitivity in 18 patients with LV hypertrophy (necropsy LV mass more than 215 g) was 100% (18 of 18 patients) and specificity was 86% (29 of 34 patients). ASE-cube LV mass correlated similarly to necropsy LV mass (r = 0.90, p less than 0.001), but systematically overestimated it (by a mean of 25%); the overestimation could be corrected by the equation: LV mass = 0.80 (ASE-cube LV mass) + 0.6 g. Use of ASE measurements in the volume correction formula systematically underestimated necropsy LV mass (by a mean of 30%). In a subset of 9 patients, 3 of whom had technically inadequate M-mode echocardiograms, 2-dimensional echocardiographic (echo) LV mass by 2 methods was also significantly related to necropsy LV mass (r = 0.68, p less than 0.05 and r = 0.82, p less than 0.01). Among other indexes of LV anatomy, only measurement of myocardial cross-sectional area was acceptably accurate for quantitation of LV mass (r = 0.80, p less than 0.001) or diagnosis of LV hypertrophy (sensitivity = 72%, specificity = 94%).(ABSTRACT TRUNCATED AT 250 WORDS)
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The mechanisms by which volatile anesthetics produce general anesthesia are unknown. Voltage-gated calcium currents in central neurons are potential target sites for general anesthetics because they are involved in the regulation of excitability and are essential for synaptic transmission. Freshly isolated rat hippocampal pyramidal neurons were studied using the whole-cell patch clamp method. Calcium currents were isolated from other voltage-activated currents by blocking sodium and potassium channels. Calcium current subtypes were studied using the specific blockers nitrendipine and omega-conotoxin GVIA. Isoflurane inhibited multiple voltage-gated calcium currents in hippocampal neurons. Isoflurane inhibited both the high- and low-voltage-activated calcium current in a clinically relevant concentration range, giving half-maximal inhibition of the peak high-voltage-activated current (measured at current maximum) at about 2% gas phase concentration, and the sustained current (measured at the end of an 800-ms depolarization) at about 1%. Isoflurane also accelerated both components of the two-component exponential decay of the high-voltage-activated current. Studies using specific channel blockers showed that the calcium current contained contributions from T, L, N, and other channels, including probably P channels. Isoflurane inhibited all of these in clinically relevant concentrations, although detailed analysis of the effects on the individual channel types was not attempted. Given the importance of calcium currents in the regulation of excitability in central neurons and the involvement of P and N channels in neurotransmitter release, this effect may represent an important action of volatile anesthetics in producing general anesthesia.