Voltage-gated sodium channels initiate action potentials in prokaryotes and in many eukaryotic cells, including vertebrate nerve and muscle. Their activation is steeply voltage-dependent, but it is unclear how the voltage sensitivity is set or whether it can be broadly shifted to positive voltages. Here we show that the voltage dependence of activation (VA) of the ancestral bacterial sodium channel NaVAb can be progressively shifted from -118 mV to +35 mV in chimeras with increasing numbers of amino acid residues from the extracellular half of the voltage sensor of human NaV1.7 channels. In a minimal chimera in which only 32 residues were transferred, we analyzed the effects of six additional mutations of conserved amino acid residues singly, in pairs, and as triple mutations. The resulting chimeric mutants exhibited a broad range of voltage sensitivity from VA=-118 mV to VA=+120 mV. Three mutations (N48K, L112A, and M119V) shifted VA to +61 mV when substituted in NaVAb itself, and substitution of two additional Cys residues in the Cys-free background of NaVAb further shifted VA to +105 mV. In these mutants, measurement of gating currents revealed that the voltage dependence of gating charge movement (VQ) shifted to positive membrane potentials as much or more than VA, confirming that the gating charges are trapped in their resting positions by these VA-shifting mutations. Our results demonstrate broadband shifting of VA and VQ of a sodium channel across a range of 240 mV and provide a toolbox of methods and constructs to analyze sodium channel structure and function in the resting state at 0 mV and in activated states at positive membrane potentials.

中文翻译：

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宽带调谐钠通道的电压依赖性

电压门控钠通道在原核生物和许多真核细胞（包括脊椎动物神经和肌肉）中启动动作电位。它们的激活与电压密切相关，但是尚不清楚如何设置电压灵敏度或是否可以将其大致转换为正电压。在这里，我们显示了在人类嵌合体中，随着人类细胞电压传感器的细胞外半部氨基酸残基数量的增加，祖先细菌性钠通道NaVAb的活化（VA）的电压依赖性可以从-118 mV逐渐转变为+35 mV。 NaV1.7频道。在仅转移了32个残基的最小嵌合体中，我们分别，成对和作为三重突变分析了保守氨基酸残基的六个其他突变的影响。所得的嵌合突变体表现出从VA = -118mV到VA = + 120mV的宽范围的电压敏感性。当用NaVAb自身取代时，三个突变（N48K，L112A和M119V）将VA移至+61 mV，并且在NaVAb无Cys的背景中取代了另外两个Cys残基，将VA移至+105 mV。在这些突变体中，对门控电流的测量表明，门控电荷移动（VQ）的电压依赖性移至正膜电位，其幅度大于或大于VA，从而证实了门控电荷被这些VA移位突变捕获在其静止位置。