Genetic disruption of voltage-gated calcium channels in psychiatric and neurological disorders

1Excitable cells can be defined as those that are able to fire an action potential in response to depolarization, or more loosely, they contain functional voltage-gated ion channels. Neurons and muscle cells are conventionally excitable, but many other cell types show oscillatory changes in voltage, dependent on calcium entry. Free intracellular Ca2+ is normally controlled at a low level in the cytoplasm (10–100 nM), by plasma membrane and endoplasmic/sarcoplasmic reticulum pumps and exchangers, as well as by mitochondrial sequestering. Voltage-gated calcium channels react to membrane potential depolarization by opening to allow calcium ions to flow down their electrochemical gradient. Ca2+ entry, particularly but not exclusively through voltage-gated calcium channels, provides an elevation of calcium ions to drive many processes. These include hormone secretion, neurotransmitter release, calcium-dependent gene transcription, and also spontaneous pacemaker activity in some neurons, muscles, and secretory cells. Ca2+ activates numerous calcium-dependent enzymes, including kinases, phosphatases, and proteases, and also activates ion channels, including Ca2+-activated K+ channels. It therefore has multiple effects on cell function depending crucially on where Ca2+ entry occurs. In skeletal muscle, the voltage-gated calcium channels link depolarization to release of internal Ca2+ from sarcoplasmic reticulum in the process of excitation-contraction coupling, and in cardiac and smooth muscle, the Ca2+ entry through voltage-gated calcium channels mediates Ca2+-induced Ca2+ release.

2Because of the key roles of voltage-gated calcium channels in processes that are so critical for cellular function, it is essential that they are inserted into the plasma membrane in a regulated manner. Muscles, secretory cells, and neurons all have specialized regions of their plasma membrane where voltage-gated calcium channels are found to be clustered in an ordered manner. The distinctive biophysical properties that are intrinsic to the different subtypes of pore-forming calcium channel subunits, as well as their regulation by auxiliary subunits and other binding proteins, ensure that the functions of voltage-gated calcium channels are customized for the different roles that they fulfil in particular subcellular locations and cells in which they occur. It also goes some way to explaining why there are so many different diseases associated, either directly or indirectly, with voltage-gated calcium channel dysfunction.

3There is currently a great deal of interest in voltage-gated calcium channels, with regard to their involvement in the genesis of a spectrum of psychiatric disorders; particularly autism spectrum disorder, schizophrenia, and bipolar disorder, and this review aims to bring together both the genetic and the available functional information.

4In order to understand how voltage-gated calcium channels may relate to neuropsychiatric diseases, it is important to be aware of the classification of these channels, their different properties and functions, as well as their distribution and interaction with other proteins, which are summarized in this section.

5The first clear evidence for more than one category of voltage-gated calcium channel was that currents underlying the calcium conductances in several cell types had distinct high- and low-voltage-activated components, which also had distinctive kinetic properties. Pharmacological tools were essential to further define these components of the calcium currents found in different neurons and muscle cells. The development of calcium channel blockers then allowed particular subtypes of voltage-gated calcium channel to be more clearly defined. The first organic blockers to be characterised were those that selectively targeted a subclass of high-voltage activated channels, prevalent in cardiac and smooth muscle. These drugs included verapamil, diltiazem, and 1,4-dihydropyridines. The channels that were sensitive to these drugs were termed L-type, particularly because the single-channel openings were of large conductance.

6Following the discovery of a number of cone shell and spider peptide toxins that selectively blocked different voltage-gated calcium channel components, further subtypes of voltage-gated calcium channel could be delineated on both physiological and pharmacological bases. In addition to the L-type channels, these were named the N-type, P/Q-type, and R-type for the high-voltage activated calcium channels, whereas the low-voltage activated channels were termed T-type (,,,,; for review, see ).

7Voltage-gated calcium channels can consist of maximally four different subunits, the pore-forming α1 subunit, as well as the auxiliary α2δ and β (and in some cases γ) subunits. The α1 subunits principally determine the kinetics, voltage dependence, single-channel conductance, and pharmacology of the voltage-gated calcium channels, although many of these properties can be modulated by the β and α2δ auxiliary subunits, which also have significant roles in voltage-gated calcium channel trafficking.

8Functional voltage-gated calcium channels are formed from one of 10 different mammalian calcium channel α1 subunit gene products, encoded by the CACNA1 genes. The nomenclature used for the channel and gene names is described here and is summarized in Fig. 1A. In the case of the CaV1.1–CaV1.4 channels (known as L-type channels), these are encoded by CACNA1S, C, D, and F, respectively. The CaV2.1–CaV2.3 channels (termed P/Q-, N-, and R-type channels) are encoded by CACNA1A, B, and E, respectively. Both the CaV1 and CaV2 channels form an α1/β/α2δ complex, co-assembling with one of four α2δ subunits (encoded by CACNA2D1–4) and one of four β subunits (encoded by CACNB1–4) (Fig. 1B). For the CaV3 channels (encoded by CACNA1G, H, and I), the α1 subunits can form functional channels alone, but may also associate with other proteins.

9All of the subunit transcripts may exhibit a number of variants, as a result of alternative splicing events. The different channel isoforms and possible combinations make for an enormous potential diversity in the properties and function of the calcium channel complexes.

10The auxiliary α2δ and β subunits also have major roles in trafficking the CaV1 and CaV2 channels to the plasma membrane and to specific domains of polarized cells, including neurons. From purification studies, the L-, P/Q-, and N-type calcium channels were all found to be associated with auxiliary β and α2δ subunits. However, the association of α2δ subunits with the calcium channel complexes was found to be relatively weak and easily disrupted, compared to the more robust interaction of the β subunits, which show a low nM affinity for interaction with the I–II linker of CaV1 and CaV2 channels. Despite this difference, both the β and α2δ subunits markedly enhance the expression and function of these channels.

11Skeletal muscle calcium channels are also found to be associated with a γ subunit (termed γ1, encoded by CACNG1), but other so-called γ subunits (encoded by CACNG2–8), do not appear to form part of cardiac or neuronal calcium channel complexes, and are better considered as transmembrane AMPA-glutamate receptor modifying proteins.

12CaV1.1 is the skeletal muscle isoform of this family and shows very low expression in brain. In contrast, CaV1.2 (α1C) encoded by CACNA1C is the predominant calcium channel in ventricular cardiac muscle and is also present in smooth muscle, many secretory cells, and throughout the brain. CaV1.3 (α1D) encoded by CACNA1D has a more restricted distribution than CaV1.2, being particularly important in sinoatrial node in the heart and in inner hair cells of the ear, although it is also present in the brain. The differential distribution and function(s) of CaV1.2 and CaV1.3 in various brain regions is discussed in detail in a recent review. CaV1.3 is activated at lower voltage thresholds than CaV1.2. CaV1.4 (α1F), encoded by CACNA1F, shows very restricted expression, mainly in the retina. The L-type channels, CaV1.2 and CaV1.3, both have a postsynaptic role to play in dendritic signalling, and in signalling to the nucleus in a process termed excitation–transcription coupling.

13Members of the CaV2 class of channels have a predominantly neuronal distribution. CaV2.1 (α1A) is the molecular equivalent of P/Q-type calcium channels and is encoded by CACNA1A. CaV2.1 channels are present throughout the brain and particularly prevalent in cerebellum. They are the predominant calcium channels involved in neurotransmission in most mature central presynaptic terminals investigated and also make up a substantial proportion of the calcium current recorded in many neuronal cell bodies, particularly Purkinje neurons. CaV2.2, or α1B, is the molecular counterpart of the N-type calcium channels and is encoded by CACNA1B. It is widely distributed throughout the central and peripheral nervous systems and is particularly important for neurotransmission early in development and also in the mature peripheral nervous system, including nociceptive pathways. CaV2.3 (α1E) is encoded by CACNA1E. It was originally described as a low-voltage activated channel, but is now understood to correspond, at least in part, to the residual R-type calcium current, present after pharmacological block of N-type, P/Q-type, and L-type channels. CaV2.3 is widely distributed in many brain regions including the hippocampus and is present both pre- and post-synaptically.

14In neurons, the CaV2 channels, particularly CaV2.1 and CaV2.2 (P/Q- and N-type calcium channels), are essential in most synapses for supplying the Ca2+ that mediates presynaptic transmitter release and there is a developmental switch towards greater reliance on CaV2.1 channels at many synapses in mature animals. CaV2.3 has been found to be differentially important for triggering spontaneous release of glutamate, although there are caveats to the use of the blocker SNX-482 to delineate the physiological roles of CaV2.3.

15The CaV3 group of channels (α1G, α1H, and α1I), encoded by CACNA1G, H, and I are the molecular counterparts of the T-type calcium channels and are more divergent with respect to their sequence from the high-voltage activated channels. CaV3 channels are widely distributed in excitable cells. In the brain, they are present in most neurons and are particularly prevalent in the thalamus. CaV3 channels (and also CaV1.3 which is relatively low-voltage activated) have important roles in neuronal excitability and pacemaker activity. As well as having a postsynaptic distribution and function, at some synapses they also have a presynaptic function to modulate, and in some cases directly mediate, transmitter release.

16CaV1 channels are inhibited by a number of L-type calcium channel blockers, many of which are in clinical use, particularly for hypertension. The most widely used are dihydropyridines, such as nifedipine. Other drugs that block L-type calcium channels, including verapamil and diltiazem, interact with overlapping high-affinity drug-binding sites on these channels. These drugs inhibit CaV1.2 more effectively than CaV1.3 or CaV1.4, partly because the drugs have a much higher affinity for the inactivated state of the channels, and CaV1.2 shows more inactivation than CaV1.3 or CaV1.4 (for review, see ). Furthermore, because vascular smooth muscle generally sits at a more depolarized membrane potential than most neurons or cardiac ventricular muscle, CaV1.2 in these cells is preferentially targeted by the voltage-dependent dihydropyridine antagonists. An additional nuance is that although these drugs can bind to the skeletal muscle calcium channel CaV1.1, the primary essential voltage-sensor function of this channel is not significantly inhibited by these calcium channel blockers.

17Members of the CaV2 class of channels are not blocked by low concentrations of dihydropyridine antagonists. CaV2.1 can be blocked by certain peptide toxins such as ω-agatoxin IVA. CaV2.2 is irreversibly inhibited by the peptide toxin, ω-conotoxin GVIA, and reversibly blocked by other related ω-conotoxins, one of which (ω-conotoxin MVIIC) is licensed for use in certain chronic pain conditions. R-type calcium current is defined as that which is present after pharmacological block of N-type channels with ω-conotoxin GVIA, block of P/Q-type channels with ω-agatoxin IVA, and block of L-type channels with a selective dihydropyridine antagonist. By this process of elimination, it is thought to be the molecular counterpart of CaV2.3 channels. R-type current can be blocked by a peptide toxin SNX 482; however this toxin also inhibits L-type channels and some K+ currents, and thus cannot readily be used to define the role of CaV2.3 channels physiologically.

18There are a number of pharmacological agents that target CaV3 channels, including mibefradil, although this compound is poorly selective and also targets other calcium channels. In contrast, TTA-A2 and TTA-P2 are selective CaV3 blockers, which are of use in defining the physiological roles of T-type channels. Z944 is also a selective T-type channel blocker, with therapeutic potential.

19Genetic analysis of disorders that do not show clear Mendelian inheritance can involve a number of different strategies. Historically, the candidate gene strategy for schizophrenia was often centred on the hypothesis that monoaminergic systems were disrupted, based on the ability of drugs (for example, chronic amphetamine), to mimic some of the symptoms of schizophrenia, and the fact that the mainstay of drug treatment of schizophrenia remains the dopamine D2 receptor antagonists. However, these approaches did not lead to useful identification of genes disrupted in these pathways, in part because, by current standards, the early candidate gene studies used very small samples, therefore they did not have adequate statistical power to detect true risk alleles even if they existed in the candidate genes tested. Nevertheless, for a few of these candidate genes (notably the gene encoding the D2 dopamine receptor, DRD2), an association has indeed now been established from large genome-wide association studies (GWAS).

20A second linkage analysis approach is aimed at identification of the genes mutated in rare families in which the disorder appears to be inherited. For example, the gene Disrupted in Schizophrenia 1 (DISC1) was identified in this way, by virtue of its association with a chromosomal translocation breakpoint. However, this type of study has had limited success with respect to understanding the aetiology of common psychiatric disorders, because of their inherently complex polygenic nature. In large resequencing studies, the hypothesis that genes, such as DISC1, contain uncommon allelic variants that are of relevance to schizophrenia has not been borne out. Nevertheless, meta-analyses have shown some consistency across linkage studies.

21Thus, aided by the rapid advances in high-throughput genotyping and sequencing technologies, other unbiased techniques are now widely employed. These include GWAS, which examine the frequencies of common genetic variants represented by single nucleotide polymorphisms (SNPs), across a large number of subjects with the disorder in question, compared to a set of matched controls. SNPs are defined as point mutations occurring with a frequency of greater than 1 in 100, and subjects can therefore be either heterozygous or homozygous for these alleles. The definition of an allele is a particular sequence at any given locus in the genome where more than one sequence is present in the population. SNPs are found throughout the genome and most are inevitably in non-coding regions. Furthermore, it is not usually known whether the implicated SNP actually represents the disease-associated alteration, or whether it is in linkage disequilibrium with the variant causing the functional effect. The effect size of individual SNPs associated with common psychiatric disorders is generally small (odds ratio <1.2). However, studies have now shown that collectively, common alleles can account for a large fraction of the genetic liability to develop psychiatric disorders. For example, one comprehensive GWAS calculated that common SNPs can collectively account for at least 32% of the variance in liability to develop schizophrenia in the population studied. GWAS have identified a number of associations, surpassing genome-wide levels of significance, in calcium channel genes for these diseases, as described below.

22Evidence that rare mutations in calcium channel genes also have an important role in the development of neuropsychiatric disorders has primarily come from whole exome sequencing studies. Whole exome sequencing is best suited for discovering rare coding mutations, such as single nucleotide variants, which can either be synonymous (not change the amino acid sequence) or non-synonymous, and either change amino acid residues (missense) or produce premature truncations by mutation to a stop codon. They may also identify deletion or insertion of one or more base pairs (indels), which cause truncation by changing the coding frame, usually resulting in introduction of a premature stop codon preceded by one or more aberrant residues. These studies can be performed in a number of different ways, either de novo mutations are identified in cases with the disorder and compared with those found in matched controls; or rare/de novo mutations in affected subjects are compared to their own unaffected parents and sibling(s) (trio/quartet studies) to identify variants associated with the condition. Since it has become clear that all humans carry a large number of rare, potentially deleterious mutations, these types of systematic controls are important, if one is to infer a causative role for such mutations in the disorder under examination. It should also be noted that gain-of-function mutations are harder to predict than loss of function mutations and therefore could be missed by any study.

23Studies investigating rare copy number variations (these are >1 kb in size and consist of large deletions, duplications, or other more complex rearrangements) have also identified calcium channel genes to be associated with some of these disorders (see below). It is likely that the functional consequence of whole gene deletion or duplication is altered gene dosage. The functional consequences of copy number variations that partially intersect a gene are more difficult to interpret, but they could have similar effects to loss of function single nucleotide variants/indels.

24Given the evidence that the spectrum of psychiatric disorders under discussion in this review all involve synaptic dysfunction, and a large number of synaptic proteins harbour disease-associated SNPs, deleterious mutations, and copy number variations, it is perhaps not surprising that voltage-gated calcium channels are one of the groups of synapse-associated proteins observed to be involved in these disorders, from many studies. This will be explored in Sections 4, 5.

25SNPs in calcium channel genes have been identified as risk alleles in a spectrum of psychiatric disorders. As described above, SNPs are point mutations occurring with a frequency >0.01. Although the identified SNP does not necessarily directly cause the biological effect, it must be in linkage disequilibrium with the causative mutation; thus the position of the associated SNP is of functional importance. Most of the associated SNPs are in introns or intergenic regions, where they may be involved in cis-regulatory elements including promoter and enhancer regions or regulation of alternative splice variant expression. cis-elements are situated within or near to the regulated gene and contain binding sites for regulatory factors required for tissue-specific and temporal expression of genes.

26The common allele associations identified in calcium channel genes, particularly those found in CACNA1C (SNP rs1006737 and other SNPs in linkage disequilibrium with this SNP), have been reproduced in multiple studies to be associated with psychiatric disorders, including schizophrenia (http://bdgene.psych.ac.cn/geneDetail.do?name=CACNA1C). For example, in a recent study, SNP rs4765905 in CACNA1C showed an association with schizophrenia with an odds ratio of 1.0944 and P = 1.2e-8. Many of these SNPs are quite common in the population, for example, at rs1006737 the SNP can be either G or A, with A being the risk variant and occurring in the general population with a frequency of about 0.33. In the most recent study, in which 108 SNPs were identified as risk factors in schizophrenia, three calcium channel gene SNPs were identified as risk factors with genome-wide levels of significance. For CACNA1C (SNP rs2007044), the odds ratio for schizophrenia was 0.912 for the common allele (P = 3.22e-18). For CACNB2 (SNP rs7893279), the odds ratio was 1.125 (P = 1.97e-12), and for CACNA1I (Chr22_39987017_D) the odds ratio was 0.930 (P = 4.725e-11). SNPs in several of these genes, particularly CACNA1C, had also been identified previously, and subsequently confirmed with greater power, in studies of people with bipolar disorder. It is important to note that some identified SNPs are associated with a specific susceptibility to either bipolar disorder or schizophrenia, whereas for two of the genes, CACNA1C and CACNB2, the SNPs were found to confer susceptibility to schizophrenia, bipolar disorder, and also to major depressive disorder. Indeed SNPs in multiple voltage-gated calcium channel subunit genes were associated with disease in this five disorder meta-analysis, including the α1 subunit genes CACNA1C, CACNA1D, CACNA1E, CACNA1S, the α2δ subunit genes CACNA2D2 and CACNA2D4, and the β subunit gene CACNB2.

27SNPs in other calcium channel genes have been found to be associated with autism spectrum disorder, in particular, the T-type calcium channel genes CACNA1G, and CACNA1I. In the latter study, 2781 parent/affected child trios were examined, and associations were considered significant if they survived correction for the 10 genes examined. Furthermore, although it is not a psychiatric disorder, variations in central pain perception and processing have been associated with an SNP in CACNA2D3.

28It has recently been demonstrated that healthy individuals exhibit multiple rare de novo or inherited neutral and disruptive mutations in coding sequences. This was well illustrated by an ion channel exome sequencing study of subjects with epilepsy, compared to controls which did not find an excess of rare potentially causative mutations in the affected individuals.

29In the largest whole exome sequencing study of schizophrenia to date, rare (less than 1 in 10,000), disruptive alleles were significantly enriched in cases, among ∼2500 genes previously implicated in schizophrenia. At the level of gene sets, disruptive alleles were enriched in voltage-gated calcium channels, with the strongest signal found for ultra-rare alleles observed only once in the sample (12 in cases, one in controls; P = 2 × 10−3, odds ratio = 8.4). Strikingly, in this study, the voltage-gated calcium channel gene subset was highly represented by CACNA1B, CACNA1C, CACNA1H, CACNA1S, CACNB4, and also CACNA2D1, CACNA2D2, and CACNA2D4. Two mutations in CACNA1C were identified; this was one of the calcium channel genes previously implicated in several studies by GWAS of both bipolar disorder and schizophrenia (see section above). Both these mutations are truncating and predicted to cause loss of function. We show, in Fig. 2, the positions of a number of the mutations in calcium channel genes, in CACNA1B, CACNA1C, CACNA1H, CACNA1S; CACNA2D1, CACNA2D2, CACNA2D4, and CACNB4, described in. All these mutations involve either frameshift, mutation to a stop codon, or splice site mutations, and from their position all are likely to cause partial or complete loss of function (Fig. 2). Given their rarity, individuals with these mutations are likely to be heterozygous and therefore carry one non-mutated allele.

30A recent study has highlighted the importance of accurate prediction of whether transcripts containing a premature stop codon will trigger nonsense-mediated mRNA decay. In the case of the truncated α1 subunits, if the mRNA was not completely degraded by nonsense-mediated decay, and if a partial protein were translated, it could potentially behave in a dominant-negative manner, as has been shown for truncated CaV2.1, CaV2.2, and CaV3 channels, and this may potentially occur for CaV2.1 in the monogenic disorder episodic ataxia 2. It should also be noted that CACNA2D4, encoding α2δ-4, implicated in (Fig. 2) is primarily expressed in the retina.

31Although not directly related to calcium channels, it is of interest that in another whole exome sequencing study in schizophrenia, disruptive mutations were identified in members of the postsynaptic density gene set, including the gene for a calcium activated protease (CAPN5). This gene encodes calpain, which can be activated in dendrites by voltage-gated calcium channels.

32Exome sequencing in individuals with autism spectrum disorder also identified an excess of de novo deleterious mutations (nonsense, splice site, and frame shifts) in cases, compared to unaffected siblings. In a recent analysis of de novo, inherited and case–control mutations identified from exome sequence of 3871 autism cases and 9937 ancestry-matched or parental controls, CACNA2D3 was implicated as a risk gene following the identification of two de novo loss of function mutations in cases and none in controls (false discovery rate <0.05). Interestingly, in a previous study of 343 families, each with a single child on the autism spectrum, and at least one unaffected sibling, a single de novo splice site mutation was found in CACNA2D3, one of many gene-disrupting mutations identified. A copy number variation study in autism also revealed a deletion in CACNA2D3. CACNA2D3 encodes α2δ-3, which is widely expressed in the brain and involved in neurotransmitter release and synaptic function.

33In the same large exome sequencing study described above that identified CACNA2D3, five mutations were also identified in CACNA1D; these included G407R (in exon 8a) and A749G, both of which were described previously and subsequently shown to cause a gain of function. Other mutations identified were A59V, which maps to an N-terminal sequence involved in Ca2+-dependent inactivation, and two C-terminal mutations (S1977L and R2021H), which are in a C-terminal proline-rich domain, involved in interaction with SHANK3.

34Furthermore, in a whole genome sequencing study of autism spectrum disorder, a rare missense mutation in CACNA1C (R1522Q) in the proximal C-terminus was identified in a proband with autism but it was also found in an unaffected sibling, and also their father (who had the cardiac disorder, Wolff Parkinson White syndrome). In this family, it was therefore unclear whether the mutation in CACNA1C was causative of autism. In another study, three rare missense variants in the coding region of CACNB2 (G167S, S197F, and F240L) were identified in autism spectrum disorder probands, and absent from controls, following exome sequencing of this gene for 259 controls and 155 probands. However, the variants showed incomplete segregation with the disorder in the families affected. Nevertheless, all three variants showed altered time-dependent inactivation of Ca2+ currents, although two mutations were associated with significantly slower inactivation, whereas the third mutation (F240L) showed increased inactivation. More recently, in an exome sequencing study of autism risk genes, investigating families where two siblings were autistic, a mutation in CACNB2 (V2D) was identified as one of relatively few genes that showed mutations in both affected siblings. The functional relevance of this mutation has not yet been studied.

35It has now been shown that there is an increased burden of microdeletions and duplications (all included in the term copy number variation) in schizophrenia but not in bipolar disorder. These copy number variations often involve multiple genes, so that it is difficult to identify with certainty the causative genes (for review, see ). For example, copy number variation in CACNA1B has been observed in subjects with disruption of the subtelomeric 9q34 region, but the rearrangements usually also involve another gene, EHMT1, and the phenotype observed is variable. In some cases, however, there was monogenic duplication of the CACNA1B gene, with a phenotype including Autism Spectrum Disorder.

36Other presynaptic markers have also been found to be implicated in this spectrum of disorders, including the synaptic scaffolding protein neurexin-1 encoded by NRXN1. Neurexins are key molecules involved in synaptic function. The α-neurexins have been found to organize active zones, by promoting the functional coupling of voltage-gated Ca2+ channels to presynaptic machinery. Neurexins are also selectively trafficked to presynaptic terminals in transport vesicles, together with other presynaptic proteins including calcium channels. Recently, rare exonic micro-deletions and other mutations in NRXN1 have been linked with schizophrenia and autism; and early GWA studies provided some evidence for common schizophrenia risk alleles existing in this gene. Although NRXN1 was not one of the 108 genes identified from the recent schizophrenia GWA study, it is of interest that this study did implicate NLGN4, encoding a neuroligin, which is a postsynaptic binding partner of neurexins.

37For autism spectrum disorder, data relating to copy number variations and deleterious single nucleotide variants have recently been combined to show convergence on a few pathways, involving chromatin modification/transcription regulation, MAP kinase/cellular signalling, and neuronal development/axon guidance. CACNA1C, CACNA1B, and CACNA1F were included in this analysis in the latter two gene networks.

38In investigating whether SNPs, rare single nucleotide variants and other genetic disruptions are associated with gain or loss of function of the implicated channel subunits, it depends on the way in which these terms are defined. Channel truncation or disruption of mRNA expression resulting from the very rare deleterious mutations described above are likely to result in loss of channel function and may even be dominant-negative, if a channel fragment is produced (Table 1). In contrast, some point mutations may increase calcium currents or reduce inactivation, which could be seen crudely as gain of function, although since tight control must be exerted over intracellular Ca2+ levels, loss of channel inactivation is also a deleterious event in neurons and other cell types (Table 1).

39Furthermore, a recent study identified a group of 19 genes implicated in schizophrenia from their previous GWAS results and showed their brain mRNA expression was correlated and further pointed out that these gene products either interact with each other or with other schizophrenia-associated gene products. Their data support a prominent role for calcium channels and associated calcium signaling pathways in the pathogenesis of schizophrenia.

40Studies on the neuropsychiatric phenotype of Timothy syndrome, resulting from CaV1.2 gain of function mutations (see Section 7.3), suggest that Cav1.2 dysfunction may operate as a risk factor in these disorders more generally. In the GWA studies described above, the association signal identified in the CACNA1C gene is in a large intron between exons 3 and 4, which contains several SNPs in linkage disequilibrium, that have been implicated in this neuropsychiatric disease spectrum. The implicated SNPs include rs1006737 and rs2007044. Recent studies on the pathophysiological consequences of these intronic risk alleles are divergent. It has been found that there is a specific interaction of an enhancer region, situated in the intron containing this SNP, with the proximal CACNA1C gene promoter, which results in the risk variant SNP being associated with reduced CACNA1C gene expression. Another recent study also reported that the CACNA1C risk alleles rs1006737 and rs1024582 are associated with decreased gene expression in post-mortem cerebellum, but not in parietal cortex. In contrast, in another study of post-mortem human brain samples from dorsolateral prefrontal cortex of control subjects, the homozygous rs1006737 risk allele (AA) was associated with a small increase in CaV1.2 mRNA. Furthermore, in a study comparing induced neurons derived from skin fibroblasts, from subjects with either AA, AG, or GG containing alleles, the presence of the homozygous rs1006737 risk allele AA also resulted in increased CACNA1C mRNA compared to those with AG or GG. In parallel, these neurons exhibited increased calcium current density for 10/11 risk allele lines analysed. However, in one of the cell lines analysed, homozygous for the risk genotype, there was lower CACNA1C mRNA levels and lower calcium current density, and thus the pattern is not completely penetrant.

41Human functional studies, examining heterozygous and homozygous individuals harbouring the risk allele for SNP rs1006737, have identified alterations in brain function, using functional magnetic resonance imaging (fMRI), associated with working memory. However, these investigations are limited by examining small numbers of subjects, and some contradictory results are reported between the studies. Interestingly, in one study, healthy carriers of the CACNA1C risk variant rs1006737 were found to have a reduction of bilateral hippocampal activation during episodic memory recall, and several other alterations. The rs1006737 risk allele was also associated in a dose-dependent manner with blunted reward responsiveness. Another recent study compared the brain activity of healthy rs1006736 risk allele carriers with matched controls. Here, a significant decrease in activity in the dorsolateral prefrontal cortex was observed, as well as a decrease the connectivity with the medial temporal lobe during working memory tasks, in those homozygous for the risk allele, which was the opposite result compared to a previous study. Although these two studies appear to be confounding, the data are actually consistent with another study that identified both increases and decreases in the capacity of working memory in bipolar and schizophrenic rs1006737 carriers, respectively. It is also consistent with studies that identified both increased and reduced expression of CACNA1C in the presence of the rs1006737 risk allele (Table 1).

42A number of other behavioural and imaging studies have also demonstrated alterations in individuals carrying the CACNA1C rs1006737 risk allele, although again these are not always consistent. An fMRI study of emotional processing in the amygdala in bipolar, schizophrenic, and healthy control carriers of the rs1006737 risk allele (AA/AG) found that the presence of the risk allele affected amygdala activity during emotional processing across all diagnostic groups. Another study showed a significant association for alleles in intron 3 of CACNA1C (both rs1006737 and the related SNP rs7959938) with increased brainstem volume. However, an effect of the rs1006737 risk allele on global grey matter volume which had been identified previously in healthy individuals could not be confirmed in this study.

43It is notable that this gene has not been implicated in the spectrum of neuropsychiatric disorders discussed above. It encodes the mainly presynaptic P/Q-type calcium channel (CaV2.1), which is primarily essential for neurotransmitter release at mature excitatory and inhibitory synapses and is also extremely important in cerebellar function, being strongly expressed in Purkinje neurons. Multiple dominant gain and loss of function mutations in CACNA1A have been described in patients, resulting in a spectrum of disorders: in particular, familial hemiplegic migraine (FHM)-1, Episodic ataxia (EA)-2, spinocerebellar ataxia (SCA)-6, and epilepsy. These diseases and their molecular basis have been described in extensive recent reviews and will not be covered in detail here. In summary, mutations in CaV2.1 causing FHM1 are thought to result in gain of function changes in CaV2.1 expression resulting in increased excitatory synaptic function and increased basal Ca2+. In contrast, the mainly truncating mutations that result in EA2 are due to loss of function, with the possibility of dominant-negative action of the mutant gene product. The mechanism of SCA6, which results from an increased CAG repeat of one C-terminal CaV2.1 splice variant, is still unclear. The question remains as to whether the pathology is due to altered CaV2.1 function or to the expanded (21–30) CAG repeat region, which is nevertheless a relatively low number of repeats that would not be deleterious in other CAG expansion diseases (for discussion, see ).

44Until recently, no mutations in CACNA1B, which encodes the mainly presynaptic neuronal N-type calcium channel CaV2.2, had been described in patients. Recently, one inherited gain of function mutation has now been described, in the outer mouth of the channel pore, which is found to be linked to a myoclonus-dystonia syndrome, although this finding has been disputed.

45This is an autosomal dominant genetic disorder affecting multiple organs, but in particular displaying cardiac defects. It is characterized primarily by prolonged QT syndrome, syndactyly, and craniofacial abnormalities, although patients also exhibit cognitive impairment, major developmental delays, and autism-like behaviours. The predominant genetic causes are de novo missense mutations, particularly in mutually exclusive exons 8 or 8a of CACNA1C, which encodes residues at the base of domain I S6, associated with the activation gate. These mutations are described as gain of function (Table 1), as they involve loss of channel inactivation, which results in elevated Ca2+ entry. In cardiomyocytes, the increased Ca2+ entry gives rise to delayed repolarization of the action potential and ventricular arrhythmias, which are the main cause of death.

46The Timothy syndrome mutations are subdivided into TS1 mutations (G406R in exon 8a) and TS2 mutations (G406R or G402S in exon 8). Tissue-specific differences in the expression of these two mutually exclusive exons result in patients with a mutation in exon 8 showing some variation in symptoms from those with a mutation in exon 8a. It might be expected that calcium channel blocking drugs would be of use in the treatment of Timothy syndrome, but the gain of function mutations are in many cases less sensitive to block, particularly by dihydropyridine antagonists, because of the loss of inactivation.

47Recently, quite a number of other rare gain of function Timothy syndrome mutations have been observed outside I S6. For example, a mutation in exon 27 (I1166T), which is predicted to be at the base of domain III S6, results in an increased window current. Also a patient with an A1473G mutation, predicted to be in domain IV S6, had a complex phenotype including seizures, and G1911R, in the C-terminus, leads to a multi-organ syndrome including seizures and developmental delay.

48Of relevance to the GWA studies described above, a patient with a G402S mutation in exon 8, for which he is a mosaic, developed bipolar disorder when aged 22. Relevant to this, heterozygous knock-in mice, expressing a human TS2 mutation (G406R) at a low level, show behavioural changes consistent with autistic-like behaviour. Furthermore, neurons derived from induced pluripotent stem cells from patients with the G406R mutation show multiple changes in gene expression, probably secondary to altered CaV1.2 function and also exhibit activity-dependent dendrite retraction.

49The CaV1.3 channels encoded by CACNA1D are L-type calcium channels which are important for pacemaker activity in the sinoatrial node and brain, as well as in hearing, as identified by a rare recessive human loss of function mutation, which gives rise to sino-atrial node dysfunction and deafness (SANDD) syndrome. Somatic gain of function mutations have been identified in CACNA1D in aldosterone producing adenomas in the adrenal zona glomerulosa. These mutations are concentrated in the pore region, the activation gate, and the voltage sensors and include mutations in exon 8a, in a striking parallel to those in Timothy syndrome. Furthermore, germline gain of function mutations were also found, identical to two of the somatic mutations (G403D and I770M), in patients with juvenile hypertension resulting from hyperaldosteronism. These patients also displayed a complex neurological syndrome including cerebral palsy.

50It is interesting that two of the mutations described above to be associated with autism spectrum disorder (G407R and A749G) were located near to the residues mutated in patients with primary aldosteronism and neurological deficits. In the brain, L-type calcium channels, including CaV1.3, play a role in the pacemaker activity in brain dopaminergic neurons, and CaV1.3 is also involved in synaptic pruning during development, two processes whose disruption might be likely to be implicated in psychiatric disorders (for review, see ). It is likely that the CACNA1D gain of function mutations resulting in autism may have a milder effect on channel function, whereas severe gain of function mutations result in more extensive disability and hyperaldosteronism (Table 1).

51Rare missense mutations in CACNA1H were found in 6 of 461 individuals with autism spectrum disorder. R212C and R902W are in domain I and domain II voltage sensors, W962C is in domain II pore, and A1874V is in the proximal C terminus. However, some of the mutations were also present in unaffected family members, indicating that the mutations are not fully penetrant. However, all these mutations reduced CaV3.2 function in an expression system. It should also be noted that gain of function mutations in CaV3.2 have been linked to childhood absence epilepsy and also to hypertension associated with hyperaldosteronism.

52The α2δ-1 protein, encoded by CACNA2D1, is highly expressed in skeletal, cardiac, and smooth muscle, as well as in the brain. Mutations in CACNA2D1 have been found to be associated with cardiac dysfunction, including Brugada and short QT syndromes. However, no central phenotypes have been identified in humans, possibly because most neurons contain more than one subtype of α2δ subunit and these proteins may have a partially interchangeable function. Nevertheless, in homozygous cacna2d1 knockout mice, a mechanosensory and pain phenotype, as well as abnormal cardiac function have been documented.

53Two human family pedigrees with recessive mutations in CACNA2D2, encoding α2δ-2, causing infantile epileptic encephalopathy have been found. The carriers had no phenotype, in agreement with the lack of phenotype in heterozygote mice lacking cacna2d2 expression, despite the homozygous knockout mice having a major ataxic and epileptic phenotype.

54β4, encoded by CACNB4, is widely expressed in the brain and is one of the main β subunits associated with neuronal calcium channels (for review, see ). Its involvement in neurological disease was first suggested by studies on the lethargic mouse strain. Furthermore, a truncating mutation (loss of 38 C-terminal residues of β4) has been found in patients with generalised epilepsy and ataxia. A particular β4 splice variant is also localised in the nucleus and it may therefore have additional functions on gene expression.

55A number of studies have been performed to examine changes in gene expression of ion channels, including calcium channel subunits, in post-mortem brains from patients with neuropsychiatric diseases. However, there are many confounding factors that affect such studies, including the fact that patients have been exposed to a variety of drugs, making it difficult to reach firm conclusions. In one study, expression of CACNA1A mRNA measured by microarray assay was down-regulated in post-mortem brains of 11 patients with bipolar disorder, although this was not confirmed by RT-PCR studies. In another study, changes in expression of some calcium channel β subunit mRNAs were observed in some brain regions, in 14 patients each with bipolar disorder, schizophrenia, or major depression, but again, confounding factors age, gender, post-mortem interval, and drug treatment make firm conclusions difficult. There are, however, several other neuropsychiatric disorders in which gene expression changes do occur, as described below.

56Fragile X syndrome is the most common inherited form of intellectual disability. Fragile X syndrome has a prevalence of 1 in 2500–4000 males and 1 in 6000–8000 females. People with Fragile X syndrome show mild to moderate cognitive dysfunction, frequently associated with autistic spectrum disorders. In addition, Fragile X syndrome patients often display peripheral autonomic and sensory symptoms, including heightened tactile sensitivity and gastrointestinal motility changes. Fragile X syndrome results from the partial or complete loss of Fragile X mental retardation protein (FMRP) expression and function. FMRP is present both in the nucleus and the cytoplasm and is a component of cytoplasmic RNA granules, where it serves both to traffic-specific mRNAs to sites of translation and to stall their translation. Loss of FMRP in fmr1 knockout mice results in dysregulation of mRNA translation and an alteration of synapse number and shape. Research has concentrated particularly on the dendritic/postsynaptic role of FMRP. Loss of FMRP results in excessive and unregulated dendritic mRNA translation. However, there is now increasing evidence for an additional presynaptic role of FMRP. Loss of presynaptic FMRP reduces functional synapse formation, decreases the size of the presynaptic active zone and synaptic vesicle number, and affects presynaptic protein levels. Granules containing FMRP have been identified in central presynaptic terminals and axons, being particularly prevalent during synapse maturation. Recent studies also describe a role for FMRP in local protein synthesis in peripheral sensory axons. Although fmr1 knockout mice exhibit normal acute nociceptive responses, they show alterations in chronic responses, both peripherally and centrally. As described above, heightened tactile sensitivity and self-injurious behaviour is observed in some Fragile X syndrome patients and this could be related to dysregulation of nocifensive behaviour.

57Several calcium channel mRNAs have been identified as FMRP targets, suggesting they would be upregulated as a result of loss of FMRP, including CACNB3 and CACNA1C, and more recently a larger array of calcium channel genes including CACNA1A, CACNA1B, CACNA1E, CACNA1G, CACNA1I, CACNB1, and CACNB3. In addition to its role as an RNA binding protein, FMRP has also been shown to associate with ion channels. FMRP has been shown to interact with, and modify the activation of, both sodium-activated K+ channels and calcium-activated K+ channels. It has also recently been shown that FMRP interacts with voltage-gated calcium channels and modulates presynaptic neurotransmitter release. Furthermore, it is relevant to mention here that rare alleles in a number of FMRP targets have been associated with schizophrenia across multiple studies.

58While changes in calcium channel gene expression have not been widely reported in neurological and psychiatric diseases, in neuropathic pain this phenomenon is widely observed following damage to peripheral nerves, which have the capacity for regeneration. Peripheral sensory nerve damage has as one of its sequelae the change in transcription of many genes, which may be either up- or down-regulated. These gene expression changes have a major role in the development and maintenance of chronic pain that long outlasts the injury. One of the many molecular consequences of experimental peripheral nerve injury is an increase in the level of α2δ-1 mRNA in damaged sensory dorsal root ganglion neurons, shown by in situ hybridization, microarray analysis, PCR, and quantitative PCR. There is also an alteration in expression of α2δ-1 splice variants, with concomitant pharmacological consequences. In contrast to α2δ-1, the mRNA for α2δ-2 and α2δ-3 has been shown to be down-regulated in rat dorsal root ganglion neurons following nerve injury.

59Sensory nerve damage may occur due to direct physical trauma, or it may be a result of poorly regulated plasma glucose in diabetes, herpes virus infection, certain chemotherapeutic drugs, and other causes. The injury-induced increase in α2δ-1 protein occurs in dorsal root ganglion somata and in their axons and central terminals in the spinal cord, as determined by Western blot and immunohistochemistry. In contrast, mRNA and protein for the main calcium channel α1 subunit involved in pain transmission (CaV2.2) is not up-regulated following sensory nerve damage, although there is a change in splicing. This leads to the possibility that up-regulated α2δ-1 enhances CaV2.2 trafficking and presynaptic function, although it may also have other functions, separate from its role as a calcium channel subunit. There is also no observed up-regulation of β subunits reported in microarray or other studies. In contrast, CACNA1D expression has been shown to be down-regulated two-fold in small dorsal root ganglions.

60Although beyond the scope of this review, the role of neuronal calcium spikes and waves during brain development and in the establishment of neuronal circuitry is the subject of extensive research. Calcium spikes are found in neurons at the earliest stages of development. These spikes involve activity that is both spontaneous and also dependent on networks. In one study, L-type calcium channels were found to be involved in thalamocortical axon outgrowth. Furthermore, it has also been observed that calcium spike activity mediates dopaminergic and GABAergic specification within the ventral suprachiasmatic nucleus of Xenopus laevis. This specification is seen to be activity-dependent and is prevented by inhibiting calcium transients. Thus, it is not surprising that subtle alterations in calcium channel function can alter neuronal connectivity, influencing cognitive functions later in life. For example, as reviewed recently, GABAergic neurotransmission in the dorsolateral prefrontal cortex of subjects with schizophrenia has been found to be reduced in a number of studies. The GABAergic neurons in question are chandelier neurons which synapse onto the axon initial segment of pyramidal neurons. How these neurons become dysfunctional in schizophrenia remains to be determined, but disruption of specific genes can have a defined effect, for example, on this subtype of cortical interneuron, which can subsequently disrupt network activity and cortical integration. Furthermore, it has been shown in Drosophila melanogaster that dendritic pruning during development occurs in a sequence of events involving calcium transients, mediated by both CaV1 and CaV2 calcium channels, which activate a calcium-activated protease (calpain) pathway. Notably, members of these three gene families (CACNA1C, CACNA1D, CAPN5) are implicated in schizophrenia from the recent GWAS and whole exome sequencing investigations described in Sections 5 and 6.

61The many genome-wide investigations carried out in the last few years have emphasized the massive polygenicity in populations, where thousands of coding and non-coding loci are polymorphic, some of which represent risk alleles for a variety of disorders, and many of which are pleiotropic, spanning a number of disorders including those reviewed here. Schizophrenia and other psychiatric conditions are found to have a high heritability. A large number of studies now indicate that the genetic component of psychiatric disease is a result of a combination of multiple fairly common alleles, represented by SNPs, each with a small effect, together with a few very rare alleles, represented by deleterious mutations and copy number variations, which might produce a relatively large increased risk in a very small subset of patients with the disorder. Thus, the genetic risk of complex phenotypes, represented by the neuropsychiatric disorders described here, is conferred by a large number of both rare and common alleles distributed across the genome. The sum of these genetic risks will interact with the many environmental risk factors that have been identified to be associated with these diseases, including cannabis consumption in the case of schizophrenia.

62The voltage-gated calcium channel genes discussed in this review, which are implicated in the aetiology of a spectrum of psychiatric syndromes from bipolar disorder through schizophrenia to autistic spectrum disorder, are one piece of a jigsaw puzzle in which synaptic proteins are strongly represented. Common and rare mutations in numerous receptors and scaffolding proteins that mediate mainly postsynaptic functions have been associated with these diseases in multiple studies. However, although the rare variant associations identified from exome sequencing studies largely involve loss of function mutations, it is much less clear whether common alleles associated with particular or multiple neuropsychiatric disorders, identified in the GWA studies described here, result in an up- or down-regulation of expression of the gene in question (Table 1). Whether the presence of particular SNPs alter the relative expression of channel splice variants, which may have substantially different properties, is another possibility that has not been explored.

63It should be noted that most of the calcium channel genes implicated in these studies do not have a primarily presynaptic function; for example, the L-type channel CaV1.2 (encoded by CACNA1C), which is strongly implicated as a risk gene across the spectrum of psychiatric disorders discussed here, is not involved in presynaptic fast transmitter release. These channels mainly play a postsynaptic modulatory role, being located on cell bodies, as well as on dendritic spines and shafts. Here, they modulate dendritic processing (see, for example, ) and have a long-range role in coupling neuronal activity to gene transcription. It is of interest that distinct roles for CaV1.2 and CaV1.3 were also found in several forms of memory, and in anxiety and depression-like behaviours in mice. It is also important to realise that some of the calcium channel genes implicated in these disorders are expressed at extremely low levels in the brain, particularly CaV1.1 (encoded by CACNA1S) and CaV1.4 (encoded by CACNA1F). It is of course possible that these transcripts have a significant but transient role during development and this could also be a fruitful area for research.

64Regarding the involvement of presynaptic calcium channels, although many calcium channel subunits have been associated with the neuropsychiatric spectrum of disorders discussed in this review, there is very little evidence for involvement of CACNA1A, encoding CaV2.1, which is the key presynaptic channel involved in neurotransmitter release at mature central synapses. In contrast, alterations in CACNA1B, encoding CaV2.2, have been identified from a number of GWA and whole exome sequencing studies. CaV2.2 is more important for synaptic transmission early in development, at least in rodents. If the same is true in humans, this may point to a neurodevelopmental role for the changes in the CaV2.2 gene, observed in the neuropsychiatric studies. In contrast, there are numerous Mendelian disorders involving CaV2.1, as described above, whereas only one family has been found possibly to have a disease-associated germline point mutation in CaV2.2, although this has been disputed.

65A well-established pathway for modulation of presynaptic calcium channels occurs via presynaptic G protein coupled receptors, with dopamine receptors being particularly relevant to psychotic disorders. It has recently been found that common alleles in the gene encoding the dopamine D2 receptor, DRD2, which is the primary target of the D2 antagonist antipsychotic drugs, have been associated with schizophrenia by GWAS. One of the primary effects of dopamine acting on this receptor (which is coupled to heterotrimeric G proteins of the Gi/Go class) is presynaptic inhibition by activation of G protein activated inwardly rectifying K+ channels, and inhibition of CaV2 calcium channels (particularly CaV2.2, but also CaV2.1 and CaV2.3). Block of D2 receptors by antipsychotic drugs would have among their effects the relief of presynaptic inhibition mediated by endogenous dopamine via these pathways. It is also worth mentioning here that the cannabinoid CB1 receptor is in the same Gi/o coupled receptor superfamily as the dopamine D2 receptor, and its activation can also mediate presynaptic inhibition.

66The other calcium channel subunits implicated in the GWA and whole exome sequencing studies described here (different β and α2δ subunits) are auxiliary and will affect both CaV1 and CaV2 families of channels. Many of the considerations described above point to the dysfunctional involvement of these channels either neurodevelopmentally, or that the disruption mainly involves postsynaptic dendritic integration. This is also consistent with these disorders as neurodevelopmental, particularly in terms of gene expression, as this can alter the balance of neurons containing particular neurotransmitters, and the synaptic contacts which they form.

67As we have attempted to summarise in Table 1, we have not been able to discern a clear consensus concerning gain or loss of function of the calcium channel gene products associated with the various disorders which form the basis of this review, but since intracellular Ca2+ is so important for cellular signalling processes, and its intracellular levels are so tightly regulated in neurons, and indeed in all cells, it is highly likely that dysregulation of these calcium channels in either direction will cause disruption of neural developmental pathways. A major challenge for the future is to translate the psychiatric genetic findings reviewed here into altered developmental and physiological function, involvement in pathology, and potential for personalised and stratified treatments for patients.