Sodium channel activation gating is affected by substitutions of voltage sensor positive charges in all four domains.
Sodium channel activation gating is affected by substitutions of voltage sensor positive charges in all four domains.
复制标题
DOI：
10.1085/jgp.110.4.391
复制
发表时间：
1997-10
期刊：
影响因子：
--
通讯作者：
Goldin AL
中科院分区：
文献类型：
--
作者：
Kontis KJ;Rounaghi A;Goldin AL
电压传感器的正电荷在激活和失活门控的大鼠脑IIA钠通道的作用进行了研究，通过突变的第二和第四个保守的正电荷在所有四个同源域的S4段。在编码钠通道α亚基的cDNA中构建电荷中和（通过谷氨酰胺取代）和电荷保守突变，所述钠通道α亚基通过在III-IV接头中掺入IFMQ 3突变而快速失活（West，J.W.，D.E. Patton，T. Scheuer，Y. Wang，A.L. Goldin和W.A.卡特拉尔。1992. Proc. Natl. Acad. Sci. USA. 89：10910-10914）。共构建了16个单突变体和2个双突变体，并分析了电压依赖性和激活和失活动力学。最显着的效果，观察到与取代的第四个正电荷在每个域。在域I或II中的第四个正电荷的中和产生了最大的位移的激活的电压依赖性，无论是在正方向。这种变化是伴随着积极的转变，活化和失活动力学的电压依赖性。结合这两种突变导致在半最大激活和门控价显着降低，以及更大的正移的电压依赖性的激活和失活动力学的一个甚至更大的正移。相反，结构域III中的第四个正电荷的中和导致了半最大激活电压的负移，而电荷保守突变导致了正移。中和结构域IV中的第四个电荷并没有改变激活的半最大电压，但保守取代产生了正移。这些数据支持这样的想法，即电荷和结构是S4电压传感器功能的决定因素。总的来说，数据支持一个工作模型，其中所有四个S4段有助于钠通道的电压依赖性激活。
The role of the voltage sensor positive charges in the activation and deactivation gating of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing (by glutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel α subunit that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III–IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. Proc. Natl. Acad. Sci. USA. 89:10910–10914.). A total of 16 single and 2 double mutants were constructed and analyzed with respect to voltage dependence and kinetics of activation and deactivation. The most significant effects were observed with substitutions of the fourth positive charge in each domain. Neutralization of the fourth positive charge in domain I or II produced the largest shifts in the voltage dependence of activation, both in the positive direction. This change was accompanied by positive shifts in the voltage dependence of activation and deactivation kinetics. Combining the two mutations resulted in an even larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger positive shifts in the voltage dependence of activation and deactivation kinetics. In contrast, neutralization of the fourth positive charge in domain III caused a negative shift in the voltage of half-maximal activation, while the charge-conserving mutation resulted in a positive shift. Neutralization of the fourth charge in domain IV did not shift the half-maximal voltage of activation, but the conservative substitution produced a positive shift. These data support the idea that both charge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segments contribute to voltage-dependent activation of the sodium channel.