Negative charges in the DIII-DIV linker of human skeletal muscle Na+ channels regulate deactivation gating.
Negative charges in the DIII-DIV linker of human skeletal muscle Na+ channels regulate deactivation gating.
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人体骨骼肌 Na 通道 DIII-DIV 连接器中的负电荷调节失活门控。
DOI：
10.1113/jphysiol.2002.033084
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Ruben,PeterC
中科院分区：
文献类型：
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作者：
Groome,JamesR;Fujimoto,Esther;Ruben,PeterC
构建了人骨骼肌钠离子通道hNav1.4的DIII-DIV连接子中D1309和EE 1314，15处的电荷反转、中和和取代突变，并在异种卵母细胞中表达。这些突变对电导、失活和失活的影响使用细胞上的宏补丁来确定。D1309 R引起电导-电压（g（V））曲线的去极化偏移，并增加表观激活价。D1309 R和EE 1314，15 RR延长了至峰值激活的时间。D1309 R引起稳态快速灭活曲线的去极化偏移，而EE 1314，15 RR引起超极化偏移并降低表观效价。在D1309或EE 1314，15下的电荷反转减慢了开放状态快速灭活并加速了闭合状态快速灭活。D1309 R加速从快速失活中恢复，而EE 1314，15 RR和EE 1314，15 QQ减慢恢复。通过从快速灭活开始至恢复的延迟来确定从灭活状态的灭活。D1309 R的恢复延迟缩短，但EE 1314，15 RR和EE 1314，15 QQ的恢复延迟延长。根据尾电流衰减的时间常数（τD）确定开放状态失活。D1309 R、D1309 E、EE 1314、15 RR和EE 1314、15 QQ使τ D减慢。我们的研究结果表明，在hNav1.4中，EE 1314，15处的负电荷簇在失活门控中起重要作用。这些和先前的发现表明，hNav1.4 DIII-DIV接头中带负电荷和带正电荷的残基簇差异性地调节快速失活的动力学。
Charge reversing, neutralizing and substituting mutations at D1309 and EE1314,15 in the DIII–DIV linker of the human skeletal muscle sodium channel hNav1.4 were constructed and expressed inXenopusoocytes. The effects of these mutations on conductance, inactivation and deactivation were determined using on‐cell macropatches. D1309R caused a depolarizing shift of the conductance–voltage (g(V)) curve and increased the apparent valency of activation. D1309R and EE1314,15RR increased time to peak activation. D1309R caused a depolarizing shift of the steady‐state fast inactivation curve, whereas EE1314,15RR produced a hyperpolarizing shift and decreased the apparent valency. Charge reversal at either D1309 or EE1314,15 slowed open‐state fast inactivation and accelerated closed‐state fast inactivation. D1309R accelerated recovery from fast inactivation, whereas EE1314,15RR and EE1314,15QQ slowed recovery. Deactivation from the inactivated state was determined by the delay in the onset to recovery from fast inactivation. Recovery delay was abbreviated for D1309R but was prolonged for EE1314,15RR and EE1314,15QQ. Open‐state deactivation was determined from the time constant of the decay (τD) of tail currents. τDwas slowed by D1309R, D1309E, EE1314,15RR and EE1314,15QQ. Our findings suggest an important role in deactivation gating in hNav1.4 for the negative cluster of charge at EE1314,15. These and previous findings suggest that clusters of negatively and positively charged residues in the hNav1.4 DIII–DIV linker differentially regulate the kinetics of fast inactivation.