Premature stop codons in a facilitating EF-hand splice variant of Ca_V2.1 cause episodic ataxia type 2

Abstract

Premature stop codons in CACNA1A, which encodes the α_1A subunit of neuronal P/Q-type (Ca_V2.1) Ca²⁺ channels, cause episodic ataxia type 2 (EA2). CACNA1A undergoes extensive alternative splicing, which contributes to the pharmacological and kinetic heterogeneity of Ca_V2.1-mediated Ca²⁺ currents. We identified three novel heterozygous stop codon mutations associated with EA2 in an alternately spliced exon (37A), which encodes part of an EF-hand motif required for Ca²⁺-dependent facilitation. One family had a C to G transversion (Y1854X). A dinucleotide deletion results in the same premature stop codon in a second family, and a further single nucleotide change leads to a different truncation (R1858X) in a de novo case of EA2. Expression studies of the Y1854X mutation revealed loss of Ca_V2.1-mediated current. Because these mutations do not affect the alternate exon 37B, these findings reveal unexpected dependence of cerebellar function on intact exon 37A-containing Ca_V2.1 channels.

Introduction

The CACNA1A gene on chromosome 19p13 codes for the pore-forming α_1A subunit of Ca_V2.1 calcium channels, also known as P/Q-type channels (Mori, 1991, Ophoff, 1996, Starr, 1991). CACNA1A undergoes extensive alternative splicing (Bourinet, 1999, Kanumilli, 2006, Mori, 1991, Ophoff, 1996, Soong, 2002, Starr, 1991, Zhuchenko, 1997) resulting in expression of several different variants of Ca_V2.1, which is expressed abundantly in the cell bodies and dendrites of cerebellar Purkinje and granule cells, and presynaptically throughout the nervous system. Splice variants contribute to pharmacological and biophysical differences among Ca_V2.1-mediated calcium currents, including but not restricted to typical P-type and Q-type behavior, as defined by sensitivity to specific toxins.

The cytoplasmic C-terminus of the Ca_V2.1 α₁-subunit contains an EF-hand domain that has been implicated in modulation of the channel by intracellular Ca²⁺-calmodulin (Liang et al., 2003). It exists in two mutually exclusive forms, determined by alternative splicing of exon 37 of CACNA1A. Both exons 37A and 37B are 97 base pairs in length and are 43% and 72% homologous on the DNA and protein level, respectively. The complete EF-hand motif is encoded by exons 36 and 37 together. Inclusion of exon 37A gives rise to a channel containing the EFa variant, which has been shown to exhibit activity-dependent enhancement of Ca²⁺ current in response to repetitive depolarization (Chaudhuri, 2004, Chaudhuri, 2007, Dunlap, 2007). Channels containing the EFb variant, which result from inclusion of exon 37B, in contrast, exhibit Ca²⁺-dependent inactivation.

Heterozygous CACNA1A mutations have been associated with a wide spectrum of episodic and progressive neurological disorders. These include familial hemiplegic migraine (FHM), spinocerebellar ataxia type 6 (SCA6) and episodic ataxia type 2 (EA2), sometimes complicated by epilepsy and mild mental retardation (Baloh, 1997, Denier, 1999, Imbrici, 2004, Jouvenceau, 2001, Ophoff, 1996, Zhuchenko, 1997). EA2 is characterized by attacks of cerebellar ataxia and interictal nystagmus. Many patients have headaches during attacks, which are often diagnosed as basilar-type migraine (Baloh et al., 1997). In addition, progressive cerebellar ataxia with gait unsteadiness, limb ataxia and dysarthria can occur (Kramer et al., 1995).

Several genetic mechanisms underlie these diseases, including missense mutations in association with various phenotypes, expansion of a CAG repeat in SCA6 and premature stop codons and splice-site mutations in association with EA2. Although it is generally agreed that EA2 arises from loss of Ca²⁺ channel function (Guida, 2001, Jen, 2001, Jouvenceau, 2001, Wappl, 2002), it remains unclear how this leads to cerebellar incoordination (Walter et al., 2006). Here we describe the clinical and functional characteristics of three previously undescribed mutations in the CACNA1A gene, all of which give rise to premature stop codons in EFa. Heterologous expression of a cDNA containing one of these stop codons reveals a non-functional α_1A subunit. Because the affected individuals are heterozygous for the mutation, which only affects splice isoforms containing exon 37A, these results argue that loss of less than 50% of Ca_V2.1 function is sufficient to cause ataxia. Furthermore, they suggest that neural circuits that rely on exon 37A-containing (EFa) splice variants, which exhibit Ca²⁺-dependent facilitation, play an important role in normal cerebellar function.