Zhang Y, Wang T, Ma A, Zhou X, Gui J, Wan H, Shi R, Huang C, Grace A A, Huang C L-H, Trump D, Zhang H, Zimmer T, Lei M
Cardiovascular Ion Channel Disease Laboratory, Department of Paediatrics, First Affiliated Hospital, Medical College of Xi'an Jiaotong University, Xi'an, China.
Acta Physiol (Oxf). 2008 Dec;194(4):311-23. doi: 10.1111/j.1748-1716.2008.01883.x. Epub 2008 Jul 24.
We compared the clinical and physiological consequences of the novel mutation R878C in a highly conserved pore residue in domain II (S5-S6) of human, hNa(v)1.5, cardiac Na(+) channels.
Full clinical evaluation of pedigree members through three generations of a Chinese family combined with SCN5A sequencing from genomic DNA was compared with patch and voltage-clamp results from two independent expression systems.
The four mutation carriers showed bradycardia, and slowed sino-atrial, atrioventricular and intraventricular conduction. Two also showed sick sinus syndrome; two had ST elevation in leads V1 and V2. Unlike WT-hNa(v)1.5, whole-cell patch-clamped HEK293 cells expressing R878C-hNa(v)1.5 showed no detectable Na(+) currents (i(Na)), even with substitution of a similarly charged lysine residue. Voltage-clamped Xenopus oocytes injected with either 0.04 or 1.5 microg microL(-1) R878C-hNa(v)1.5 cRNA similarly showed no i(Na), yet WT-hNa(v)1.5 cRNA diluted to 0.0004-0.0008 ng microL(-1)resulted in expression of detectable i(Na). i(Na) was simply determined by the amount of injected WT-hNa(v)1.5: doubling the dose of WT-hNa(v)1.5 cRNA doubled i(Na). i(Na) amplitudes and activation and inactivation characteristics were similar irrespective of whether WT-hNa(v)1.5 cRNA was given alone or combined with equal doses of R878C-hNa(v)1.5 cRNA therefore excluding dominant negative phenotypic effects. Na(+) channel function in HEK293 cells transfected with R878C-hNa(v)1.5 was not restored by exposure to mexiletine (200 microM) and lidocaine (100 microM). Fluorescence confocal microscopy using E3-Nav1.5 antibody demonstrated persistent membrane expression of both WT and R878C-hNa(v)1.5. Modelling studies confirmed that such i(Na) reductions reproduced the SSS phenotype.
Clinical consequences of the novel R878C mutation correlate with results of physiological studies.
我们比较了人类心脏钠通道hNa(v)1.5结构域II（S5-S6）中一个高度保守的孔道残基处的新型突变R878C的临床和生理后果。
对一个中国家族的三代成员进行全面临床评估，并结合对基因组DNA进行SCN5A测序，将其与两个独立表达系统的膜片钳和电压钳结果进行比较。
四名突变携带者表现出心动过缓，以及窦房、房室和心室内传导减慢。两人还表现出病态窦房结综合征；两人在V1和V2导联出现ST段抬高。与野生型hNa(v)1.5不同，表达R878C-hNa(v)1.5的全细胞膜片钳HEK293细胞即使替换了带相同电荷的赖氨酸残基，也未检测到钠电流（i(Na)）。注射0.04或1.5μg/μL R878C-hNa(v)1.5 cRNA的电压钳非洲爪蟾卵母细胞同样未显示i(Na)，而野生型hNa(v)1.5 cRNA稀释至0.0004-0.0008 ng/μL时可检测到i(Na)的表达。i(Na)仅由注射的野生型hNa(v)1.5的量决定：野生型hNa(v)1.5 cRNA剂量加倍，i(Na)也加倍。无论野生型hNa(v)1.5 cRNA单独给予还是与等量的R878C-hNa(v)1.5 cRNA联合给予，i(Na)幅度以及激活和失活特性均相似，因此排除了显性负性表型效应。用美西律（200μM）和利多卡因（100μM）处理转染了R878C-hNa(v)1.5的HEK293细胞，钠通道功能未恢复。使用E3-Nav1.5抗体的荧光共聚焦显微镜显示野生型和R878C-hNa(v)1.5均持续在细胞膜表达。建模研究证实，这种i(Na)降低重现了病态窦房结综合征的表型。
新型R878C突变的临床后果与生理学研究结果相关。
