14-3-3 Is a regulator of the cardiac voltage-gated sodium channel Nav1.5
14-3-3 Is a regulator of the cardiac voltage-gated sodium channel Nav1.5
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DOI：
10.1161/01.res.0000229244.97497.2c
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发表时间：
2006-06-23
影响因子：
20.1
通讯作者：
Baro, Isabelle
中科院分区：
文献类型：
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作者：
Allouis, Marie;Le Bouffant, Francoise;Baro, Isabelle
电压敏感的Na+通道Na(v)1.5在心脏动作电位的产生和传播中起着至关重要的作用，其功能障碍可促进心律失常。该通道参与了一个包含调控蛋白的大分子复合体。因此，从生理学和病理学的角度来看，调节其生物合成、定位、活性和/或降解的因素都是非常有趣的。利用酵母2杂交筛选，我们发现了一个新的合作伙伴14-3-3 eta，它与Na(v)1.5细胞质I间域相互作用。在表达重组Na(v)1.5的COS-7细胞和小鼠心肌细胞中，通过14-3-3和全长Na(v)1.5的共免疫沉淀证实了这种相互作用。利用免疫细胞化学，我们还发现14-3-3和Nav1.5在插入椎间盘上共定位。我们使用全细胞膜片钳结构测试了Na(v)1.5和14-3-3 eta之间的功能联系。共表达Na(v)1.5的β 1亚基和14-3-3 eta诱导Na+电流失活曲线发生负移，失活恢复延迟，但激活曲线和电流密度没有变化。通过过表达与14-3-3 eta相互作用的Na(v)1.5胞质I域，使失活后的恢复归一化。r56,60a 14-3-3 - eta显性负突变体也得到逆转，表明14-3-3的二聚化是当前调控所需要的。计算机模拟表明，14-3-3缺失可能对心电恢复特性产生促心律失常的影响。基于这些发现，我们提出14-3-3蛋白是心脏Na+通道的一个新组成部分，作为心脏Na+电流调节的辅助因子。
The voltage-sensitive Na+ channel Na(v)1.5 plays a crucial role in generating and propagating the cardiac action potential and its dysfunction promotes cardiac arrhythmias. The channel takes part into a large molecular complex containing regulatory proteins. Thus, factors that modulate its biosynthesis, localization, activity, and/or degradation are of great interest from both a physiological and pathological standpoint. Using a yeast 2-hybrid screen, we unveiled a novel partner, 14-3-3 eta, interacting with the Na(v)1.5 cytoplasmic I interdomain. The interaction was confirmed by coimmunoprecipitation of 14-3-3 and full-length Na(v)1.5 both in COS-7 cells expressing recombinant Na(v)1.5 and in mouse cardiac myocytes. Using immunocytochemistry, we also found that 14-3-3 and Nav1.5 colocalized at the intercalated discs. We tested the functional link between Na(v)1.5 and 14-3-3 eta using the whole-cell patch-clamp configuration. Coexpressing Na(v)1.5, the beta 1 subunit and 14-3-3 eta induced a negative shift in the inactivation curve of the Na+ current, a delayed recovery from inactivation, but no changes in the activation curve or in the current density. The negative shift was reversed, and the recovery from inactivation was normalized by overexpressing the Na(v)1.5 cytoplasmic I interdomain interacting with 14-3-3 eta. Reversal was also obtained with the dominant negative R56,60A 14-3-3 eta mutant, suggesting that dimerization of 14-3-3 is needed for current regulation. Computer simulations suggest that the absence of 14-3-3 could exert proarrhythmic effects on cardiac electrical restitution properties. Based on these findings, we propose that the 14-3-3 protein is a novel component of the cardiac Na+ channel acting as a cofactor for the regulation of the cardiac Na+ current.