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. 2015 Dec 29:8:89.
doi: 10.1186/s13041-015-0180-4.

A mutation in the low voltage-gated calcium channel CACNA1G alters the physiological properties of the channel, causing spinocerebellar ataxia

Hiroyuki Morino  1 , Yukiko Matsuda  2 , Keiko Muguruma  3 , Ryosuke Miyamoto  4 , Ryosuke Ohsawa  5 , Toshiyuki Ohtake  6 , Reiko Otobe  7 , Masahiko Watanabe  8 , Hirofumi Maruyama  9 , Kouichi Hashimoto  10 , Hideshi Kawakami  11
Affiliations

A mutation in the low voltage-gated calcium channel CACNA1G alters the physiological properties of the channel, causing spinocerebellar ataxia

Hiroyuki Morino et al. Mol Brain. .

Abstract

Background: Spinocerebellar ataxia (SCA) is a genetically heterogeneous disease. To date, 36 dominantly inherited loci have been reported, and 31 causative genes have been identified.

Results: In this study, we analyzed a Japanese family with autosomal dominant SCA using linkage analysis and exome sequencing, and identified CACNA1G, which encodes the calcium channel CaV3.1, as a new causative gene. The same mutation was also found in another family with SCA. Although most patients exhibited the pure form of cerebellar ataxia, two patients showed prominent resting tremor in addition to ataxia. CaV3.1 is classified as a low-threshold voltage-dependent calcium channel (T-type) and is expressed abundantly in the central nervous system, including the cerebellum. The mutation p.Arg1715His, identified in this study, was found to be located at S4 of repeat IV, the voltage sensor of the CaV3.1. Electrophysiological analyses revealed that the membrane potential dependency of the mutant CaV3.1 transfected into HEK293T cells shifted toward a positive potential. We established induced pluripotent stem cells (iPSCs) from fibroblasts of the patient, and to our knowledge, this is the first report of successful differentiation from the patient-derived iPSCs into Purkinje cells. There was no significant difference in the differentiation status between control- and patient-derived iPSCs.

Conclusions: To date, several channel genes have been reported as causative genes for SCA. Our findings provide important insights into the pathogenesis of SCA as a channelopathy.

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Figures

Fig. 1
Fig. 1
Identification of a mutation in CACNA1G causing SCA. a Pedigree charts of families 1 and 2. Arrows indicate the probands. Filled and open symbols represent affected and unaffected individuals, respectively. Genotypes of the variant c.5144G > A are shown under the number of samples. Asterisks indicate the patients used for exome sequencing. b, c Results of linkage analysis. Arrows indicate the positions of CACNA1G. d Sanger sequencing to confirm the CACNA1G variant. The reference nucleotide G is overlapped with variant nucleotide A in the mutant sample. e Structure of CaV3.1 encoded by CACNA1G. The star indicates the position of the identified mutation. The mutation was located in the segment 4 (S4) of the fourth repeat. f Conservation at the location of the mutation. The nucleotide and amino acid sequences are completely conserved among vertebrates. g Haplotype analysis. From the result of SNP genotyping, the haplotypes of both families around the CACNA1G gene coincided for 360 kb
Fig. 2
Fig. 2
Changes in electrophysiological properties. a Representative traces of T-type VDCC currents recorded from HEK293T cells expressing wild-type (upper) or mutant (lower) CaV3.1. Holding potential was -60 mV. Voltage steps were applied after a hyperpolarizing prepulse to -100 mV (duration = 500 ms). b Peak current-voltage plots of VDCC currents of wild-type (blue, n = 9) and mutant (red, n = 12) CaV3.1. c Relative conductance-voltage plots. Each data point was calculated from data in B. The black line is a fit using the Boltzmann equation (see Methods). d Steady state inactivation-voltage plots. The voltage step to -30 mV was preceded by incremental hyperpolarizing pulses (duration = 300 ms). Data were obtained from the same cells shown in B and C. The black line is a fit using the Boltzmann equation (see Methods). e The decay time constants of inactivation of the Ca2+ current plotted against voltage steps. f The 10–90 % rise times of Ca2+ currents plotted against voltage steps. Data are presented as the mean ± SEM. Statistical significance was assessed by Mann-Whitney U-test. **p < 0.01, *p < 0.05
Fig. 3
Fig. 3
iPSC-derived Purkinje cells. Immunostaining of iPSC-derived Purkinje cells on co-culture days 26. GRID2 was expressed in the L7+ dendritic spines. The scale bars represent 500 μm (upper panels) and 100 μm (lower panels)
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