Cardiovascular and Genetic Medicine Research Groups, School of Biomedicine, University of Manchester, Manchester, United Kingdom.
PLoS One. 2010 Jun 7;5(6):e10985. doi: 10.1371/journal.pone.0010985.
To identify molecular mechanisms underlying SCN5A-related sick sinus syndrome (SSS), a rare type of SSS, in parallel experiments we elucidated the electrophysiological properties and the cell surface localization of thirteen human Na(v)1.5 (hNa(v)1.5) mutant channels previously linked to this disease.
METHODOLOGY/PRINCIPAL FINDINGS: Mutant hNa(v)1.5 channels expressed by HEK293 cells and Xenopus oocytes were investigated by whole-cell patch clamp and two-microelectrode voltage clamp, respectively. HEK293 cell surface biotinylation experiments quantified the fraction of correctly targeted channel proteins. Our data suggested three distinct mutant channel subtypes: Group 1 mutants (L212P, P1298L, DelF1617, R1632H) gave peak current densities and cell surface targeting indistinguishable from wild-type hNa(v)1.5. Loss-of-function of these mutants resulted from altered channel kinetics, including a negative shift of steady-state inactivation and a reduced voltage dependency of open-state inactivation. Group 2 mutants (E161K, T220I, D1275N) gave significantly reduced whole-cell currents due to impaired cell surface localization (D1275N), altered channel properties at unchanged cell surface localization (T220I), or a combination of both (E161K). Group 3 mutant channels were non-functional, due to an almost complete lack of protein at the plasma membrane (T187I, W1421X, K1578fs/52, R1623X) or a probable gating/permeation defect with normal surface localisation (R878C, G1408R).
CONCLUSIONS/SIGNIFICANCE: This study indicates that multiple molecular mechanisms, including gating abnormalities, trafficking defects, or a combination of both, are responsible for SCN5A-related familial SSS.
为了确定 SCN5A 相关病态窦房结综合征（SSS）的分子机制，我们在平行实验中阐明了先前与该疾病相关的 13 个人源 Na(v)1.5（hNa(v)1.5）突变通道的电生理特性和细胞表面定位。
方法/主要发现：通过全细胞膜片钳和双电极电压钳分别研究了在 HEK293 细胞和非洲爪蟾卵母细胞中表达的突变 hNa(v)1.5 通道。HEK293 细胞表面生物素化实验定量了正确靶向的通道蛋白的分数。我们的数据表明存在三种不同的突变通道亚型：第 1 组突变体（L212P、P1298L、DelF1617、R1632H）的峰值电流密度和细胞表面靶向与野生型 hNa(v)1.5 无明显差异。这些突变体的功能丧失是由于通道动力学的改变，包括稳态失活的负移和开放状态失活的电压依赖性降低。第 2 组突变体（E161K、T220I、D1275N）由于细胞表面定位受损（D1275N）、在不变的细胞表面定位时改变通道特性（T220I）或两者的组合（E161K）导致全细胞电流显著降低。第 3 组突变体通道由于几乎完全缺乏质膜上的蛋白（T187I、W1421X、K1578fs/52、R1623X）或具有正常表面定位的可能门控/渗透性缺陷（R878C、G1408R）而无功能。
这项研究表明，多种分子机制，包括门控异常、运输缺陷或两者的组合，导致 SCN5A 相关家族性 SSS。
