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Mutations in the CACNA1A gene are associated with episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6). CACNA1A encodes the α-subunit of the P/Q-type calcium channel or CaV2.1, which is highly enriched in the cerebellum. It is one of the main channels linked to synaptic transmission throughout the human central nervous system. Here, we compare recent advances in the understanding of the genetic changes that underlie EA2 and SCA6 and what these new findings suggest about the mechanism of the disease.
The CaV2.1 calcium channel belongs to the superfamily of voltage-gated calcium channels. Its other designation, P/Q-type channel, refers to the cell types from which its constituent currents were originally isolated; the ‘P’ stands for Purkinje and ‘Q’ for granule cells of the cerebellum. The α-subunit (Figure 1), encoded by CACNA1A, is associated with β and α2δ auxiliary subunits that are thought to help in trafficking and anchoring the principal subunit to the cell membrane and also modulate the biophysical properties of the channel . CACNA1A undergoes extensive alternative splicing resulting in CaV2.1 channels with different properties [2-5]. These splice variants are differentially expressed throughout the central nervous system (CNS) and serve to adjust the biophysical parameters of the channel to its role in the various cell types.
The CACNA1A gene maps to chromosome 19p13 . It is widely expressed throughout the CNS; however, in keeping with its original identification, this gene is expressed at a particularly high level in Purkinje and granule cells of the cerebellum. In much of the CNS, CaV2.1 channels are highly expressed pre-synaptically , where they couple calcium influx to vesicular exocytosis in fast neurotransmission. However, in Purkinje cells, CaV2.1 channels serve an additional post-synaptic role in coordinating AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor activation with voltage-dependent calcium influx .
Dominant mutations in CACNA1A underlie at least three allelic diseases (Table 1). A large number of different point mutations (both nonsense and missense) have been shown to cause episodic ataxia type 2 (EA2), an acetazolamide-responsive disorder characterised by paroxysmal attacks of midline cerebellar disturbance manifesting as ataxia, imbalance, vomiting, oscillopsia, and interictal nystagmus [9-15]. Attacks last from hours to days, and triggers include stress and intercurrent infection. Several other EA syndromes have been described (reviewed in ); mutations in KCNA1 - the gene that encodes the KV1.1 potassium channel - underlie EA1, which is characterised by brief attacks of ataxia (lasting minutes) with interictal myokymia [17-20]. Interestingly, mutations in CACNB4, the gene that encodes one of the accessory β-subunits of CaV2.1, also cause a form of EA .
EA2 mutations that result in premature stop codons are likely to generate a non-functional truncated peptide or trigger nonsense-mediated mRNA decay. Functional characterisation of missense mutations in CACNA1A has demonstrated that mutations associated with EA2 result in a loss of CaV2.1 channel function [22,23]. In addition, mutant subunits may disrupt the membrane trafficking of wild-type channels .
In contrast to EA2, spinocerebellar ataxia type 6 (SCA6) is a ‘pure’ progressive cerebellar syndrome that results, not from point mutations, but from an abnormal polyglutamine expansion in the channel's carboxyl-terminal domain, which is present in only certain splice isoforms of the CACNA1A mRNA . Although changes in channel kinetics have been observed [26,27], the pathogenic mechanism of SCA6 is poorly understood.
Direct sequencing of CACNA1A in a number of patients with clinical EA2 often fails to identify causative point mutations. However, the last year has witnessed a further step forward in understanding the genetic basis of EA2. Veneziano and colleagues  identified new 5′ and 3′ regions in the CACNA1A gene, including a gene promoter region and a new final exon 48, both of which harboured mutations in patients with EA. Furthermore, the mutation spectrum has expanded with the findings of large deletions and duplications in CACNA1A in affected individuals. Previously, nonsense and missense mutations accounted for most cases of EA2. Recently, methods such as MLPA (multiplex ligation-dependent probe amplification) and QMPSF (quantitative multiplex polymerase chain reaction of short fluorescent fragments) have demonstrated large-scale CACNA1A gene rearrangements in patients with EA2 [29,30]. This finding is particularly important for those patients with clinical EA2 in whom sequencing of CACNA1A fails to identify a point mutation.
EA2 is an autosomal dominant disease, and because large deletions in CACNA1A are not likely to produce functional transcripts, it is likely that reduced channel density in the cerebellar circuit (possibly in Purkinje cells, where these channels have been shown to play a central role) is sufficient to cause episodes of ataxia. Moreover, the recent observation that nonsense mutations located within a well-known alternatively spliced exon (exon 37A)  can cause EA2 hints at a significant role of CaV2.1 channels containing exon 37A in the cerebellum and underpins the importance of calcium channel splicing in disease causation.
While increasing evidence points to a loss of robust CaV2.1 expression in the cerebellum and haploinsufficency as the underlying mechanism of EA2, calcium channel dysfunction may not be at the root of SCA6. In support of this view, the expanded CAG repeat in the SCA6 knock-in mouse does not appear to affect CaV2.1 function , indicating that the polyglutamine repeat itself may have a cytotoxic effect on the cell. It has recently been suggested that cerebellar dysfunction in a related polyglutamine repeat SCA (SCA2) may arise from aberrant activation of type 1 inositol 1,4,5-trisphosphate receptors (ITPRs) in Purkinje cells by the glutamine tracts themselves . If activation of the ITPRs is the mechanism of polyglutamine repeat SCAs, then SCA6 may be a result of the relative abundance of P/Q channels in Purkinje cells, rather than specific properties of the channels themselves.
Despite advances in the genetic basis of EA2, many questions remain unanswered. From a mechanistic point of view, the paroxysmal nature of the neurological symptoms arising from mutations in CACNA1A remains a mystery. In addition, the physiological basis of how attacks are precipitated by stress or indeed relieved by acetazolamide still evades explanation. Answers to some of these questions may await the development of neuronal expression systems. This will allow the study of how genetic variation in the calcium channel gene affects the biophysical properties of CaV2.1 in its physiological environment, especially with respect to synaptic transmission and dendritic depolarisation. A recent insight into this approach came from Heeroma and colleagues , who demonstrated that KV1.1 mutations in EA1 exerted differential effects on excitation (rheobase) and synaptic transmission when expressed in neurons.
Although the polyglutamine tract in SCA6 is considered to confer a toxic gain of function, the exact roles of the alternative long- and short-splice variants of CACNA1A in Purkinje cells need to be defined. This may lead to a better understanding of how the expanded CAG repeat in SCA6 results in disease. An additional conundrum is why many spontaneous mutations in CACNA1A in mice result in a 3 Hz spike and wave epilepsy [35,36], whereas in humans, although a few mutations have been shown to co-segregate with EA2 and seizures [37,38], most deletions and truncations in the gene are associated with pure EA2 rather than absence epilepsy. Mutations of this gene appear to give two distinct phenotypes in humans and rodents. Understanding how these phenotypes are associated with different roles of CaV2.1 channels in human and rodent tissues may be an important step in untangling the molecular pathogenesis of seizures and ataxia.
The authors thank the Wellcome Trust, Medical Research Council (G0601943) and Action Research for financial support.
The electronic version of this article is the complete one and can be found at: http://f1000.com/reports/b/2/4
The authors declare that they have no competing interests.