Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen.
Ann Neurol. 2012 Dec;72(6):870-80. doi: 10.1002/ana.23700.
To identify the causative gene for the neurodegenerative disorder spinocerebellar ataxia type 19 (SCA19) located on chromosomal region 1p21-q21.
Exome sequencing was used to identify the causal mutation in a large SCA19 family. We then screened 230 ataxia families for mutations located in the same gene (KCND3, also known as Kv4.3) using high-resolution melting. SCA19 brain autopsy material was evaluated, and in vitro experiments using ectopic expression of wild-type and mutant Kv4.3 were used to study protein localization, stability, and channel activity by patch-clamping.
We detected a T352P mutation in the third extracellular loop of the voltage-gated potassium channel KCND3 that cosegregated with the disease phenotype in our original family. We identified 2 more novel missense mutations in the channel pore (M373I) and the S6 transmembrane domain (S390N) in 2 other ataxia families. T352P cerebellar autopsy material showed severe Purkinje cell degeneration, with abnormal intracellular accumulation and reduced protein levels of Kv4.3 in their soma. Ectopic expression of all mutant proteins in HeLa cells revealed retention in the endoplasmic reticulum and enhanced protein instability, in contrast to wild-type Kv4.3 that was localized on the plasma membrane. The regulatory β subunit Kv channel interacting protein 2 was able to rescue the membrane localization and the stability of 2 of the 3 mutant Kv4.3 complexes. However, this either did not restore the channel function of the membrane-located mutant Kv4.3 complexes or restored it only partially.
KCND3 mutations cause SCA19 by impaired protein maturation and/or reduced channel function.
鉴定位于 1p21-q21 染色体区域的神经退行性疾病脊髓小脑共济失调 19 型（SCA19）的致病基因。
使用外显子组测序鉴定一个大型 SCA19 家族的致病突变。然后，我们使用高分辨率熔解筛选了位于同一基因（KCND3，也称为 Kv4.3）中的 230 个共济失调家族的突变。评估了 SCA19 脑尸检材料，并通过膜片钳技术使用野生型和突变型 Kv4.3 的异位表达进行体外实验，以研究蛋白质定位、稳定性和通道活性。
我们在电压门控钾通道 KCND3 的第三细胞外环检测到 T352P 突变，该突变与我们原始家族的疾病表型共分离。我们在另外 2 个共济失调家族中发现了另外 2 个通道孔（M373I）和 S6 跨膜域（S390N）的新错义突变。T352P 小脑尸检材料显示严重的浦肯野细胞退化，其胞体中 Kv4.3 的异常细胞内积累和蛋白水平降低。所有突变蛋白在 HeLa 细胞中的异位表达均显示在内质网中滞留和增强蛋白不稳定性，而野生型 Kv4.3 则定位于质膜上。调节β亚基 Kv 通道相互作用蛋白 2 能够挽救 2 种突变 Kv4.3 复合物的膜定位和稳定性。然而，这要么不能恢复膜定位的突变 Kv4.3 复合物的通道功能，要么只能部分恢复。
KCND3 突变通过蛋白成熟受损和/或通道功能降低导致 SCA19。
