Neuronal voltage-gated calcium channels generate rapid, transient intracellular calcium signals in response to membrane depolarization. Neuronal CaV channels regulate a range of cellular functions and are implicated in a variety of neurological and psychiatric diseases including epilepsy, Parkinson’s disease, chronic pain, schizophrenia, and bipolar disorder. Each mammalian Cacna1 gene has the potential to generate tens to thousands of CaV channels by alternative pre-mRNA splicing, a process that adds fine granulation to the pool of CaV channel structures and functions. The precise composition of CaV channel splice isoform mRNAs expressed in each cell are controlled by cell-specific splicing factors. The activity of splicing factors are in turn regulated by molecules that encode various cellular features, including cell-type, activity, metabolic states, developmental state, and other factors. The cellular and behavioral consequences of individual sites of CaV splice isoforms are being elucidated, as are the cell-specific splicing factors that control splice isoform selection. Altered patterns of alternative splicing of CaV pre-mRNAs can alter behavior in subtle but measurable ways, with the potential to influence drug efficacy and disease severity. This article is part of a Special Issue entitled: Calcium channels.
Splicing factor; Disease; Chronic pain; Morphine; Synaptic transmission; G protein coupled receptor; Mu-opioid receptor
Alternative splicing of low-voltage-activated T-type calcium channels contributes to the molecular and functional diversity mediating complex network oscillations in the normal brain. Transcript scanning of the human CACNA1G gene has revealed the presence of 11 regions within the coding sequence subjected to alternative splicing, some of which enhance T-type current. In mouse models of absence epilepsy, elevated T-type calcium currents without clear increases in channel expression are found in thalamic neurons that promote abnormal neuronal synchronization. To test whether enhanced T-type currents in these models reflect pathogenic alterations in channel splice isoforms, we determined the extent of alternative splicing of mouse Cacna1g transcripts and whether evidence of altered transcript splicing could be detected in mouse absence epilepsy models.
Transcript scanning of the murine Cacna1g gene detected 12 regions encoding alternative splice isoforms of Cav3.1/α1G T-type calcium channels. Of the 12 splice sites, six displayed homology to the human CACNA1G splice sites, while six novel mouse-specific splicing events were identified, including one intron retention, three alternative acceptor sites, one alternative donor site, and one exon exclusion. In addition, two brain region-specific alternative splice patterns were observed in the cerebellum. Comparative analyses of brain regions from four monogenic absence epilepsy mouse models with altered thalamic T-type currents and wildtype controls failed to reveal differences in Cacna1g splicing patterns.
The determination of six novel alternative splice sites within the coding region of the mouse Cacna1g gene greatly expands the potential biophysical diversity of voltage-gated T-type channels in the mouse central nervous system. Although alternative splicing of Cav3.1/α1G channels does not explain the enhancement of T-type current identified in four mouse models of absence epilepsy, post-transcriptional modification of T-type channels through this mechanism may influence other developmental neurological phenotypes.
The calcium channel CACNA1A gene encodes the pore-forming, voltage-sensitive subunit of the voltage-dependent calcium Ca(v)2.1 type channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine, episodic ataxia 2 and spinocerebellar ataxia type 6. The mouse homologue, Cacna1a, is associated with the tottering, Cacna1atg, mutant series. Here we describe two new missense mutant alleles, Cacna1atg-4J and Cacna1aTg-5J. The Cacna1atg-4J mutation is a valine to alanine mutation at amino acid 581, in segment S5 of domain II. The recessive Cacna1atg-4J mutant exhibited the ataxia, paroxysmal dyskinesia and absence seizures reminiscent of the original tottering mouse. The Cacna1atg-4J mutant also showed altered activation and inactivation kinetics of the Ca(v)2.1 channel, not previously reported for other tottering alleles. The semi-dominant Cacna1aTg-5J mutation changed a conserved arginine residue to glutamine at amino acid 1252 within segment S4 of domain III. The heterozygous mouse was ataxic and homozygotes rarely survived. The Cacna1aTg-5J mutation caused a shift in both voltage activation and inactivation to lower voltages, showing that this arginine residue is critical for sensing Ca(v)2.1 voltage changes. These two tottering mouse models illustrate how novel allelic variants can contribute to functional studies of the Ca(v)2.1 calcium channel.
tottering alleles; Ca(v)2.1 calcium channels; semi-dominant mutation
The CACNA1C gene (alpha 1C subunit of the L-type voltage-gated calcium channel) has been identified as a risk gene for both bipolar disorder and schizophrenia but the mechanism of association has not been explored.
To identify the neural system mechanism that explains the genetic association between the CACNA1C gene and psychiatric illness, using neuroimaging and human brain expression.
We used BOLD fMRI to measure brain activation in circuitries related to bipolar disorder and schizophrenia by comparing CACNA1C genotype groups in healthy subjects. We tested the effect of genotype on mRNA levels of CACNA1C in post-mortem human brain. A case-control analysis was used to determine the association of CACNA1C genotype and schizophrenia.
National Institutes of Health Clinical Center
Healthy Caucasian men and women participated in the fMRI study. Post-mortem samples from normal human brains were used for the brain expression study. Patients with schizophrenia and healthy subjects were used in the case-control analysis.
Main Outcome Measures
BOLD fMRI, mRNA levels in post-mortem brain samples, and genetic association with schizophrenia
The risk associated single nucleotide polymorphism (SNP rs1006737) in CACNA1C predicted increased hippocampal activity during emotional processing (puncorr=0.001, pFDR=0.052, Z=3.20) and increased prefrontal activity during executive cognition (puncorr=2.8e-05, pFDR=0.011, Z=4.03). The risk SNP also predicted increased expression of CACNA1C mRNA in human brain (p=0.0017). CACNA1C was associated with schizophrenia in our case-control sample (OR 1.77, p=0.026).
The risk associated SNP in CACNA1C maps to circuitries implicated in genetic risk for both bipolar disorder and schizophrenia. Its effects in human brain expression implicate a molecular and neural systems mechanism for the clinical genetic association.
Spinocerebellar ataxia type 6 (SCA6) is an inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the CaV2.1 voltage-gated calcium channel subunit (CACNA1A). There is currently no treatment for this debilitating disorder and thus a pressing need to develop preventative therapies. RNA interference (RNAi) has proven effective at halting disease progression in several models of spinocerebellar ataxia (SCA), including SCA types 1 and 3. However, in SCA6 and other dominantly inherited neurodegenerative disorders, RNAi-based strategies that selectively suppress expression of mutant alleles may be required. Using a CaV2.1 mini-gene reporter system, we found that pathogenic CAG expansions in CaV2.1 enhance splicing activity at the 3′end of the transcript, leading to a CAG repeat length-dependent increase in the levels of a polyQ-encoding CaV2.1 mRNA splice isoform and the resultant disease protein. Taking advantage of this molecular phenomenon, we developed a novel splice isoform-specific (SIS)-RNAi strategy that selectively targets the polyQ-encoding CaV2.1 splice variant. Selective suppression of transiently expressed and endogenous polyQ-encoding CaV2.1 splice variants was achieved in a variety of cell-based models including a human neuronal cell line, using a new artificial miRNA-like delivery system. Moreover, the efficacy of gene silencing correlated with effective intracellular recognition and processing of SIS-RNAi miRNA mimics. These results lend support to the preclinical development of SIS-RNAi as a potential therapy for SCA6 and other dominantly inherited diseases.
The Cacna1fnob2 mouse is reported to be a naturally occurring null mutation for the Cav1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1fnob2 mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Cav1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Cav1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Cav1.4wt or Cav1.4nob2 cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1fnob2 mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1fnob2 mice was generally similar to that of wild type mice. These results suggest the Cacna1fnob2 mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Cav1.4 protein, albeit at reduced levels, and the functional Cav1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1fnob2 mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts.
Mutations in the CACNA1F gene encoding the L-type calcium channel pore-forming Cav1.4 (α1F) subunit in humans result in an incomplete form of congenital stationary night blindness (CSNB2) with residual photoreceptor function. It has been postulated that this residual function, at least in part, may be mediated by another L-type calcium channel subunit, Cav1.3 (α1D), expressed within cone photoreceptors. However, the expression of the calcium channel Cav1.3 (α1D) subunit within photoreceptors remains debatable due to discrepancies among the immunohistochemical studies reported in the literature. In order to get around the innate complications of utilizing unproven antibodies and to shed light on this discussion, we investigated the mRNA expression profile for the Cav1.3 (α1D) subunit in the mouse retina.
In situ hybridization was performed on wild type mouse retinal sections with two independent sets of digoxigenin-11-UTP-labeled Cav1.3 (α1D)-specific sense and antisense cRNA probes. The two probe sets employed correspond to two distinct regions of the Cav1.3 (α1D) subunit mRNA, each encoding a different fragment of the Cav1.3 (α1D) polypeptide. In situ hybridization of wild type mouse brain sections with these same probes was performed as an additional control for specificity.
Abundant L-type calcium channel Cav1.3 (α1D) subunit mRNA expression was confirmed in most cells of the outer nuclear layer using two independent Cav1.3 (α1D)-specific antisense cRNA probes, confirming expression in rod photoreceptors. Cav1.3 (α1D) mRNA expression was also observed within most cells of the inner nuclear layer and ganglion cell layers using these same antisense cRNA probes. No labeling of tissue was observed using either sense cRNA probe. In situ detection of concentrated Cav1.3 (α1D) mRNA expression within the hippocampus and Purkinje and granule cells of the cerebellum of wild type mouse brain with these same probes confirmed specificity of the probes.
Our finding of expression of the L-type calcium channel Cav1.3 (α1D) subunit mRNA in rods substantiates the possibility that this pore-forming subunit may be a competent component of channels mediating the residual photoreceptor responses observed in mutant mice lacking functional Cav1.4 (α1F) subunits and in humans with CSNB2. Furthermore, the combined observations of abundant expression of Cav1.3 (α1D) mRNA in wild type rods and the large reduction in the transmission of photoreceptor responses in mice lacking Cav1.4 (α1F) raises the possibility that Cav1.3 (α1D) protein expression levels, localization, or functioning might be concomitantly altered by disruption of the Cav1.4 (α1F) subunit in rods. To date, no studies of Cav1.3 (α1D) mRNA nor protein expression levels or localization in cacna1f mutant mice or humans with CSNB2 have been published. Our findings warrant such studies to address the abovementioned possibilities. Finally, the observation of Cav1.3 (α1D) mRNA expression in multiple retinal cell types suggests the potential for a broader role for this L-type calcium channel subunit in overall functioning of the normal retina than previously appreciated. We therefore suggest that lesions in either the gene encoding the L-type calcium channel Cav1.3 (α1D) subunit or other molecules that interact with and regulate it may underlie one or more retinopathies with currently unidentified molecular etiologies.
Light-dependent conductance changes of voltage-gated Cav1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Cav1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Cav1.4 channels, but some mutations alter the channels’ gating properties and, presumably, disturb Ca2+ influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14–17) in a longitudinal study up to eight months of age. In ΔEx14–17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca2+ responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca2+ response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14–17 mutants.
Voltage-gated Ca2+ (Cav) channels control neuronal functions including neurotransmitter release and gene expression. The Cacna1a gene encodes the α1 subunit of the pore-forming Cav2.1 channel. Mice with mutations in this gene form useful tools for defining channel functions. The recessive ataxic tottering-6j strain that was generated in the Neuroscience Mutagenesis Facility at The Jackson Laboratory has a mutation in the Cacna1a gene. However, the effect of this mutation has not been investigated in detail. In this study, mutation analysis shows a base substitution (C-to-A) in the consensus splice acceptor sequence linked to exon 5, which results in the skipping of exon 5 and the splicing of exon 4 directly to exon 6. The effect of this mutation is expected to be severe as the expressed α1 subunit protein lacks a significant part of the S4–S5 linker, S5, and part of S5–S6 linker in domain I. Tottering-6j mice display motor dysfunctions in the footprint, rotating rod, and hind-limb extension tests. Although cytoarchitecture of the mutant brains appears normal, tyrosine hydroxylase was persistently expressed in cerebellar Purkinje cells in the adult mutant mice. These results indicate that tottering-6j is a useful model for functional studies of the Cav2.1 channel.
One of the most consistent genetic findings to have emerged from bipolar disorder genome wide association studies (GWAS) is with CACNA1C, a gene that codes for the α1C subunit of the Cav1.2 voltage-dependent L-type calcium channel (LTCC). Genetic variation in CACNA1C have also been associated with depression, schizophrenia, autism spectrum disorders, as well as changes in brain function and structure in control subjects who have no diagnosable psychiatric illness. These data are consistent with a continuum of shared neurobiological vulnerability between diverse—Diagnostic and Statistical Manual (DSM) defined—neuropsychiatric diseases. While involved in numerous cellular functions, Cav1.2 is most frequently implicated in coupling of cell membrane depolarization to transient increase of the membrane permeability for calcium, leading to activation and, potentially, changes in intracellular signaling pathway activity, gene transcription, and synaptic plasticity. Cav1.2 is involved in the proper function of numerous neurological circuits including those involving the hippocampus, amygdala, and mesolimbic reward system, which are strongly implicated in psychiatric disease pathophysiology. A number of behavioral effects of LTCC inhibitors have been described including antidepressant-like behavioral actions in rodent models. Clinical studies suggest possible treatment effects in a subset of patients with mood disorders. We review the genetic structure and variation of CACNA1C, discussing relevant human genetic and clinical findings, as well as the biological actions of Cav1.2 that are most relevant to psychiatric illness.
Calcium channel; CACNA1C; Genome wide association study; Bipolar disorder; Depression; Schizophrenia; Psychiatric genetics
The CACNA1A gene, encoding the voltage-gated calcium channel subunit α1A, is involved in pre- and postsynaptic Ca2+ signaling, gene expression, and several genetic neurological disorders. We found that CACNA1A employs a novel strategy to directly coordinate a gene expression program, using a bicistronic mRNA bearing a cryptic internal ribosomal entry site (IRES). The first cistron encodes the well-characterized α1A subunit. The second expresses a newly-recognized transcription factor, α1ACT, that coordinates expression of a program of genes involved in neural and Purkinje cell development. α1ACT also contains the polyglutamine (polyQ) tract that, when expanded, causes spinocerebellar ataxia type 6 (SCA6). When expressed as an independent polypeptide, α1ACT, bearing an expanded polyQ tract, lacks transcription factor function and neurite outgrowth properties, causes cell death in culture, and leads to ataxia and cerebellar atrophy in transgenic mice. Suppression of CACNA1A IRES function in SCA6 may be a potential therapeutic strategy.
Presynaptic voltage-gated calcium CaV2.2 channels play a privileged role in spinal level sensitization following peripheral nerve injury. Direct and indirect inhibitors of CaV2.2 channel activity in spinal dorsal horn are analgesic in chronic pain states. CaV2.2 channels represent a family of splice isoforms that are expressed in different combinations according to cell-type. A pair of mutually exclusive exons in the CaV2.2 encoding Cacna1b gene, e37a and e37b, differentially influence morphine analgesia. In mice that lack exon e37a, which is enriched in nociceptors, the analgesic efficacy of intrathecal morphine against noxious thermal stimuli is reduced. Here we ask if sequences unique to e37a influence: the development of abnormal thermal and mechanical sensitivity associated with peripheral nerve injury; and the actions of two other classes of analgesics that owe part or all of their efficacy to CaV2.2 channel inhibition. We find that: i) the analgesic efficacy of morphine, but not ziconotide or gabapentin, is reduced in mice lacking e37a, ii) the induction and maintenance of behaviors associated with sensitization that accompany peripheral nerve injury, do not require e37a-specific sequence, iii) intrathecal morphine, but not ziconotide or gabapentin analgesia to thermal stimuli is significantly lower in wild-type mice after peripheral nerve injury, iv) the analgesic efficacy of ziconotide and gabapentin to mechanical stimuli is reduced following nerve injury, and iv) intrathecal morphine analgesia to thermal stimuli in mice lacking e37a is not further reduced by peripheral nerve injury. Our findings show that the analgesic action of morphine, but not ziconotide or gabapentin, to thermal stimuli is linked to which Cacna1b exon, e37a or e37b, is selected during alternative pre-mRNA splicing.
Voltage-gated calcium channels; Neuropathic pain; Alternative splicing; Morphine; Ziconotide; Gabapentin; Nociception; Analgesia; Spared nerve injury
Voltage-dependent L-type Ca2+ (CaV1.2) channels are the principal Ca2+ entry pathway in arterial myocytes. CaV1.2 channels regulate multiple vascular functions and are implicated in the pathogenesis of human disease, including hypertension. However, the molecular identity of CaV1.2 channels expressed in myocytes of myogenic arteries that regulate vascular pressure and blood flow is unknown. Here, we cloned CaV1.2 subunits from resistance size cerebral arteries and demonstrate that myocytes contain a novel, cysteine rich N terminus that is derived from exon 1 (termed “exon 1c”), which is located within CACNA1C, the CaV1.2 gene. Quantitative PCR revealed that exon 1c was predominant in arterial myocytes, but rare in cardiac myocytes, where exon 1a prevailed. When co-expressed with α2δ subunits, CaV1.2 channels containing the novel exon 1c-derived N terminus exhibited: 1) smaller whole cell current density, 2) more negative voltages of half activation (V1/2,act) and half-inactivation (V1/2,inact), and 3) reduced plasma membrane insertion, when compared with channels containing exon 1b. β1b and β2a subunits caused negative shifts in the V1/2,act and V1/2,inact of exon 1b-containing CaV1.2α1/α2δ currents that were larger than those in exon 1c-containing CaV1.2α1/α2δ currents. In contrast, β3 similarly shifted V1/2,act and V1/2,inact of currents generated by exon 1b- and exon 1c-containing channels. β subunits isoform-dependent differences in current inactivation rates were also detected between N-terminal variants. Data indicate that through novel alternative splicing at exon 1, the CaV1.2 N terminus modifies regulation by auxiliary subunits. The novel exon 1c should generate distinct voltage-dependent Ca2+ entry in arterial myocytes, resulting in tissue-specific Ca2+ signaling.
To examine changes in inner retinal function of nob2 mice, expressing a null mutation in Cacna1f encoding the CaV1.4 subunit of voltage-dependent calcium channels. CACNA1F mutations underlie one form of incomplete X-linked congenital stationary night blindness (CSNB2). In addition to a loss of dark-adapted (rod-driven) visual sensitivity, electroretinogram (ERG) b-waves and oscillatory potentials (OPs) are decreased in CSNB2 patients.
ERGs were recorded under dark-and light-adapted conditions from the corneal surface of nob2 mice, WT littermates and nob4 mice. ERG frequency spectra were calculated by fast Fourier transform (FFT). A FFT-based high-pass filter was used to derive OP waveforms.
Under dark-adapted conditions, the dominant frequency of the OPs varied between 90 to 120 Hz in WT mice. In WT mice, OP frequency first increased with flash intensity and then decreased at the highest flash levels while overall OP amplitude increased monotonically with increasing flash intensity. In response to low stimulus flashes, reliable OPs were not obtained from nob2 mice. OPs were only seen at stimulus intensities at or above −1.8 log cd s/m2, where they occurred at a lower frequency range (70–90 Hz) than for WT mice. When flash stimuli were superimposed against a steady rod-desensitizing adapting field, the amplitude and frequency of WT OPs increased with flash intensity above 0.4 log cd s/m2. In comparison to WT results, cone-mediated OPs obtained from nob2 mice were smaller in amplitude, of lower frequency and had delayed implicit times. We compared the extent to which OPs and the b-wave were reduced in nob2 mice, by normalizing to the results obtained from WT mice. In comparison to the b-wave, the OPs were relatively spared, under both dark- and light-adapted conditions.
In nob2 mice, rod- and cone-driven OPs are reduced in amplitude and occur at a lower frequency range. Since CaV1.4 is expressed in both the inner and outer plexiform layers, these changes are likely to reflect reduced transmission from photoreceptors to bipolar cells as well as alterations in inner retinal function. That the OPs were better preserved than b-waves suggests that inner retinal pathways may be reorganized in response to the decreased bipolar cell response in nob2 mice.
Oscillatory potentials; Electroretinogram; nob2; Mice; Frequency spectrum; Voltage dependent calcium channel
Individual differences in the sensitivity to fentanyl, a widely used opioid analgesic, lead to different proper doses of fentanyl, which can hamper effective pain treatment. Voltage-activated Ca2+ channels (VACCs) play a crucial role in the nervous system by controlling membrane excitability and calcium signaling. Cav2.3 (R-type) VACCs have been especially thought to play critical roles in pain pathways and the analgesic effects of opioids. However, unknown is whether single-nucleotide polymorphisms (SNPs) of the human CACNA1E (calcium channel, voltage-dependent, R type, alpha 1E subunit) gene that encodes Cav2.3 VACCs influence the analgesic effects of opioids. Thus, the present study examined associations between fentanyl sensitivity and SNPs in the human CACNA1E gene in 355 Japanese patients who underwent painful orofacial cosmetic surgery, including bone dissection. We first conducted linkage disequilibrium (LD) analyses of 223 SNPs in a region that contains the CACNA1E gene using genomic samples from 100 patients, and a total of 13 LD blocks with 42 Tag SNPs were observed within and around the CACNA1E gene region. In the preliminary study using the same 100 genomic samples, only the rs3845446 A/G SNP was significantly associated with perioperative fentanyl use among these 42 Tag SNPs. In a confirmatory study using the other 255 genomic samples, this SNP was also significantly associated with perioperative fentanyl use. Thus, we further analyzed associations between genotypes of this SNP and all of the clinical data using a total of 355 samples. The rs3845446 A/G SNP was associated with intraoperative fentanyl use, 24 h postoperative fentanyl requirements, and perioperative fentanyl use. Subjects who carried the minor G allele required significantly less fentanyl for pain control compared with subjects who did not carry this allele. Although further validation is needed, the present findings show the possibility of the involvement of CACNA1E gene polymorphisms in fentanyl sensitivity.
Genetic variability of the major subunit (CACNA1E) of the voltage-dependent Ca2+ channel CaV2.3 is associated to risk of type 2 diabetes, insulin resistance and impaired insulin secretion in nondiabetic subjects. The aim of the study was to test whether CACNA1E common variability affects beta cell function and/or insulin sensitivity in patients with newly diagnosed type 2 diabetes.
In 595 GAD-negative, drug naïve patients (mean±SD; age: 58.5±10.2 yrs; BMI: 29.9±5 kg/m2, HbA1c: 7.0±1.3) with newly diagnosed type 2 diabetes we: 1. genotyped 10 tag SNPs in CACNA1E region reportedly covering ∼93% of CACNA1E common variability: rs558994, rs679931, rs2184945, rs10797728, rs3905011, rs12071300, rs175338, rs3753737, rs2253388 and rs4652679; 2. assessed clinical phenotypes, insulin sensitivity by the euglycemic insulin clamp and beta cell function by state-of-art modelling of glucose/C-peptide curves during OGTT. Five CACNA1E tag SNPs (rs10797728, rs175338, rs2184945, rs3905011 and rs4652679) were associated with specific aspects of beta cell function (p<0.05−0.01). Both major alleles of rs2184945 and rs3905011 were each (p<0.01 and p<0.005, respectively) associated to reduced proportional control with a demonstrable additive effect (p<0.005). In contrast, only the major allele of rs2253388 was related weakly to more severe insulin resistance (p<0.05).
In patients with newly diagnosed type 2 diabetes CACNA1E common variability is strongly associated to beta cell function. Genotyping CACNA1E might be of help to infer the beta cell functional phenotype and to select a personalized treatment.
We have positionally cloned and characterized a new calcium channel auxiliary subunit, α2δ-2 (CACNA2D2), which shares 56% amino acid identity with the known α2δ-1 subunit. The gene maps to the critical human tumor suppressor gene region in chromosome 3p21.3, showing very frequent allele loss and occasional homozygous deletions in lung, breast, and other cancers. The tissue distribution of α2δ-2 expression is different from α2δ-1, and α2δ-2 mRNA is most abundantly expressed in lung and testis and well expressed in brain, heart, and pancreas. In contrast, α2δ-1 is expressed predominantly in brain, heart, and skeletal muscle. When co-expressed (via cRNA injections) with α1B and β3 subunits in Xenopus oocytes, α2δ-2 increased peak size of the N-type Ca2+ currents 9-fold, and when co-expressed with α1C or α1G subunits in Xenopus oocytes increased peak size of L-type channels 2-fold and T-type channels 1.8-fold, respectively. Anti-peptide antibodies detect the expression of a 129-kDa α2δ-2 polypeptide in some but not all lung tumor cells. We conclude that the α2δ-2 gene encodes a functional auxiliary subunit of voltage-gated Ca2+ channels. Because of its chromosomal location and expression patterns, CACNA2D2 needs to be explored as a potential tumor suppressor gene linking Ca2+ signaling and lung, breast, and other cancer pathogenesis. The homologous location on mouse chromosome 9 is also the site of the mouse neurologic mutant ducky (du), and thus, CACNA2D2 is also a candidate gene for this inherited idiopathic generalized epilepsy syndrome.
The fine-tuning of neuronal excitability relies on a tight control of Ca2+ homeostasis. The low voltage-activated (LVA) T-type calcium channels (Cav3.1, Cav3.2 and Cav3.3 isoforms) play a critical role in regulating these processes. Despite their wide expression throughout the central nervous system, the implication of T-type Cav3.2 isoform in brain functions is still poorly characterized. Here, we investigate the effect of genetic ablation of this isoform in affective disorders, including anxiety, cognitive functions as well as sensitivity to drugs of abuse. Using a wide range of behavioral assays we show that genetic ablation of the cacna1h gene results in an anxiety-like phenotype, whereas novelty-induced locomotor activity is unaffected. Deletion of the T-type channel Cav3.2 also triggers impairment of hippocampus-dependent recognition memories. Acute and sensitized hyperlocomotion induced by d-amphetamine and cocaine are dramatically reduced in T-type Cav3.2 deficient mice. In addition, the administration of the T-type blocker TTA-A2 prevented the expression of locomotor sensitization observed in wildtype mice. In conclusion, our data reveal that physiological activity of this specific Ca2+ channel is required for affective and cognitive behaviors. Moreover, our work highlights the interest of T-type channel blockers as therapeutic strategies to reverse drug-associated alterations.
T-type Ca2+ channels; anxiety; memory and learning; psychostimulants; drugs of abuse; spontaneous behavior
The Cacna1a gene encodes the α1A subunit of voltage-gated CaV2.1 Ca2+ channels that are involved in neurotransmission at central synapses. CaV2.1-α1-knockout (α1KO) mice, which lack CaV2.1 channels in all neurons, have a very severe phenotype of cerebellar ataxia and dystonia, and usually die around postnatal day 20. This early lethality, combined with the wide expression of CaV2.1 channels throughout the cerebellar cortex and nuclei, prohibited determination of the contribution of particular cerebellar cell types to the development of the severe neurobiological phenotype in Cacna1a mutant mice. Here, we crossed conditional Cacna1a mice with transgenic mice expressing Cre recombinase, driven by the Purkinje cell-specific Pcp2 promoter, to specifically ablate the CaV2.1-α1A subunit and thereby CaV2.1 channels in Purkinje cells. Purkinje cell CaV2.1-α1A-knockout (PCα1KO) mice aged without difficulties, rescuing the lethal phenotype seen in α1KO mice. PCα1KO mice exhibited cerebellar ataxia starting around P12, much earlier than the first signs of progressive Purkinje cell loss, which appears in these mice between P30 and P45. Secondary cell loss was observed in the granular and molecular layers of the cerebellum and the volume of all individual cerebellar nuclei was reduced. In this mouse model with a cell type-specific ablation of CaV2.1 channels, we show that ablation of CaV2.1 channels restricted to Purkinje cells is sufficient to cause cerebellar ataxia. We demonstrate that spatial ablation of CaV2.1 channels may help in unraveling mechanisms of human disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s12311-011-0302-1) contains supplementary material, which is available to authorized users.
P/Q-type Ca2+ channels; Conditional; Cell-specific knockout; Cacna1a; Ataxia
It is well established that idiopathic generalized epilepsies (IGEs) show a polygenic origin and may arise from dysfunction of various types of voltage- and ligand-gated ion channels. There is an increasing body of literature implicating both high and low voltage-activated (HVA and LVA) calcium channels and their ancillary subunits in IGEs. Cav2.1 (P/Q-type) calcium channels control synaptic transmission at presynaptic nerve terminals, and mutations in the gene encoding the Cav2.1 α1 subunit (CACNA1A) have been linked to absence seizures in both humans and rodents. Similarly, mutations and loss of function mutations in ancillary HVA calcium channel subunits known to coassemble with Cav2.1 result in IGE phenotypes in mice. It is important to note that in all these mouse models with mutations in HVA subunits there is a compensatory increase in thalamic LVA currents, which likely leads to the seizure phenotype. In fact, gain of function mutations have been identified in Cav3.2 (an LVA or T-type calcium channel encoded by the CACNA1H gene) in patients with congenital forms of IGEs, consistent with increased excitability of neurons as a result of enhanced T-type channel function. Here we provide a broad overview of the roles of voltage-gated calcium channels, their mutations, and how they might contribute to the river that terminates in epilepsy.
calcium channel; P/Q-type channels; T-type channels; epilepsy; seizures
Congenital heart block (CHB) is a transplacentally acquired autoimmune disease associated with anti-Ro/SSA and anti-La/SSB maternal autoantibodies and is characterized primarily by atrioventricular (AV) block of the fetal heart. This study aims to investigate whether the T-type calcium channel subunit α1G may be a fetal target of maternal sera autoantibodies in CHB.
We demonstrate differential mRNA expression of the T-type calcium channel CACNA1G (α1G gene) in the AV junction of human fetal hearts compared to the apex (18–22.6 weeks gestation). Using human fetal hearts (20–22 wks gestation), our immunoprecipitation (IP), Western blot analysis and immunofluorescence (IF) staining results, taken together, demonstrate accessibility of the α1G epitope on the surfaces of cardiomyocytes as well as reactivity of maternal serum from CHB affected pregnancies to the α1G protein. By ELISA we demonstrated maternal sera reactivity to α1G was significantly higher in CHB maternal sera compared to controls, and reactivity was epitope mapped to a peptide designated as p305 (corresponding to aa305–319 of the extracellular loop linking transmembrane segments S5–S6 in α1G repeat I). Maternal sera from CHB affected pregnancies also reacted more weakly to the homologous region (7/15 amino acids conserved) of the α1H channel. Electrophysiology experiments with single-cell patch-clamp also demonstrated effects of CHB maternal sera on T-type current in mouse sinoatrial node (SAN) cells.
Taken together, these results indicate that CHB maternal sera antibodies readily target an extracellular epitope of α1G T-type calcium channels in human fetal cardiomyocytes. CHB maternal sera also show reactivity for α1H suggesting that autoantibodies can target multiple fetal targets.
The mouse mutant ducky and its allele ducky2J represent a model for absence epilepsy characterized by spike-wave seizures, and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the α2δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2du2J mutation results in a two base-pair deletion in the coding region and a complete loss of α2δ-2 protein. Here we show that du2J/du2J mice have a 30% reduction in somatic calcium current, and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C du2J/du2J PCs show no spontaneous intrinsic activity. Du2J/du2J mice also have alterations in the cerebellar expression of several genes related to PC function. At P21 there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du2J/+ mice have a marked reduction in α2δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However, du2J/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α2δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α2δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma.
calcium channel; mouse mutant; α2δ subunit; mutation; spontaneous firing; Purkinje cell
Calcium channel blockers (CCBs) are widely used to treat cardiovascular diseases such as hypertension, angina pectoris, hypertrophic cardiomyopathy, and supraventricular tachycardia. CCBs selectively inhibit the inward flow of calcium ions through voltage-gated calcium channels, particularly Cav1.2, that are expressed in the cardiovascular system. Changes to the molecular structure of Cav1.2 channels could affect sensitivity of the channels to blockade by CCBs. Recently, extensive alternative splicing was found in Cav1.2 channels that generated wide phenotypic variations. Cardiac and smooth muscles express slightly different, but functionally important Cav1.2 splice variants. Alternative splicing could also modulate the gating properties of the channels and giving rise to different responses to inhibition by CCBs. Importantly, alternative splicing of Cav1.2 channels may play an important role to influence the outcome of many cardiovascular disorders. Therefore, the understanding of how alternative splicing impacts Cav1.2 channels pharmacology in various diseases and different organs may provide the possibility for individualized therapy with minimal side effects.
Low voltage-activated T-type calcium (Ca) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct T-type Ca channel isoforms can be generated by alternative splicing from each of three different T-type genes (CaV3.1, CaV3.2, CaV3.3), although it remains to be described whether specific splice variants are associated with developmental states and pathological conditions. We aimed to identify and functionally characterize CaV3.2 T-type Ca channel alternatively spliced variants from newborn animals and to compare with adult normotensive and spontaneously hypertensive rats (SHR). DNA sequence analysis of full-length CaV3.2 cDNA generated from newborn heart tissue identified ten major regions of alternative splicing, the more common variants of which were analyzed by quantitative real-time PCR (qRT-PCR) and also subject to functional examination by whole-cell patch clamp. The main findings are that: (1) cardiac CaV3.2 T-type Ca channels are subject to considerable alternative splicing, (2) there is preferential expression of CaV3.2(−25) splice variant channels in newborn rat heart with a developmental shift in adult heart that results in approximately equal levels of expression of both (+25) and (−25) exon variants, (3) in the adult stage of hypertensive rats there is both an increase in overall CaV3.2 expression and a shift towards expression of CaV3.2(+25) containing channels as the predominant form and (4) alternative splicing confers a variant-specific voltage-dependent facilitation of CaV3.2 channels. We conclude that CaV3.2 alternative splicing generates significant T-type Ca channel structural and functional diversity with potential implications relevant to cardiac developmental and pathophysiological states.
voltage-dependent facilitation; alternative splicing; T-type calcium channel; hypertension; cardiac hypertrophy
The β subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain β subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of β isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual β variants in specific neuronal functions. In the present study, an alternatively spliced β4 subunit lacking the variable N terminus (β4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length β4 variants (β4a and β4b), β4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of β4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (β4-null) mice individually reconstituted with β4a, β4b, and β4e. Notably, the number of genes regulated by each β4 splice variant correlated with the rank order of their nuclear-targeting properties (β4b > β4a > β4e). Together, these findings support isoform-specific functions of β4 splice variants in neurons, with β4b playing a dual role in channel modulation and gene regulation, whereas the newly detected β4e variant serves exclusively in calcium-channel-dependent functions.
Ca2+ channel; Cacnb4; CaV2.1; cerebellar granule cells; hippocampal neurons; lethargic mice