Relative resistance to slow inactivation of human cardiac Na+ channel hNav1.5 is reversed by lysine or glutamine substitution at V930 in D2-S6.
Relative resistance to slow inactivation of human cardiac Na+ channel hNav1.5 is reversed by lysine or glutamine substitution at V930 in D2-S6.
复制标题
D2-S6 中 V930 处的赖氨酸或谷氨酰胺取代可逆转对人心脏 Na 通道 hNav1.5 缓慢失活的相对抵抗力。
DOI：
10.1152/ajpcell.00377.2007
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发表时间：
2007
期刊：
影响因子：
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通讯作者：
O'Reilly,JohnP
中科院分区：
文献类型：
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作者：
Chancey,JessicaHotard;Shockett,PennyE;O'Reilly,JohnP
跨膜片段6涉及电压门控Na+通道（Navs）的缓慢失活（SI）。为了进一步研究其作用并了解不同Navisoforms的SI表型之间的差异，我们分析了人心脏hNav1.5的几个结构域2-片段6（D2-S6）突变体，其对SI具有相对抗性。通过瞬时HEK细胞转染和全细胞Na+电流的膜片钳记录来检查突变体。用赖氨酸（K）取代包括N927 K、V930 K和L931 K。我们发现N927 K和L931 K从短（100 ms）去极化恢复到0 mV与野生型相当，而V930 K的恢复是延迟的和双指数的，表明快速进入缓慢失活状态。与野生型hNav1.5的抗性表型相比，SI方案证实了V930 K的增强的SI表型（快速发育、超极化稳态、缓慢恢复）。这种增强在N927 K或L931 K中未发现，表明V930 K中的效应是位点特异性的。在V930处取代的谷氨酰胺（Q）也表现出类似于V930 K的增强的SI表型。因此，K或Q取代消除了hNav1.5对SI的抗性。在V930处的丙氨酸（A）或半胱氨酸（C）取代不显示SI的增强，并且事实上，V930 A和V930 C以及L931 K表现出对SI的抗性，这证明特定氨基酸（例如，大小、疏水性）差异地影响SI门控。因此，D2-S6中的V930似乎是hNav1.5中SI门控的重要结构决定因素。我们认为，涉及D2-S6的构象变化是Navs中SI的关键组成部分，这可能是由SI表型的其他亚型特异性决定因素在亚型之间进行差异调节。
Transmembrane segment 6 is implicated in slow inactivation (SI) of voltage-gated Na+channels (Navs). To further study its role and understand differences between SI phenotypes of different Navisoforms, we analyzed several domain 2-segment 6 (D2-S6) mutants of the human cardiac hNav1.5, which is relatively resistant to SI. Mutants were examined by transient HEK cell transfection and patch-clamp recording of whole cell Na+currents. Substitutions with lysine (K) included N927K, V930K, and L931K. We show recovery from short (100 ms) depolarization to 0 mV in N927K and L931K is comparable to wild type, whereas recovery in V930K is delayed and biexponential, suggesting rapid entry into a slow-inactivated state. SI protocols confirm enhanced SI phenotype (rapid development, hyperpolarized steady state, slowed recovery) for V930K, contrasting with the resistant phenotype of wild-type hNav1.5. This enhancement, not found in N927K or L931K, suggests that the effect in V930K is site specific. Glutamine (Q) substituted at V930 also exhibits an enhanced SI phenotype similar to that of V930K. Therefore, K or Q substitution eliminates hNav1.5 resistance to SI. Alanine (A) or cysteine (C) substitution at V930 shows no enhancement of SI, and in fact, V930A and V930C, as well as L931K, exhibit a resistance to SI, demonstrating that characteristics of specific amino acids (e.g., size, hydrophobicity) differentially affect SI gating. Thus V930 in D2-S6 appears to be an important structural determinant of SI gating in hNav1.5. We suggest that conformational change involving D2-S6 is a critical component of SI in Navs, which may be differentially regulated between isoforms by other isoform-specific determinants of SI phenotype.