Role of arginine residues on the S4 segment of the Bacillus halodurans Na+ channel in voltage-sensing
Role of arginine residues on the S4 segment of the Bacillus halodurans Na+ channel in voltage-sensing
复制标题
DOI：
10.1007/s00232-004-0701-z
复制
发表时间：
2004-09-01
影响因子：
2.4
通讯作者：
Sato, C
中科院分区：
文献类型：
--
作者：
Chahine, M;Pilote, S;Sato, C
耐盐芽孢杆菌的单域电压门控钠通道（NaChBac）由六个跨膜区段（S1-S6）组成，所述跨膜区段包括侧接区段S5和S6的成孔区和由区段S4组成的电压敏感元件。为了研究S4片段在NaChBac通道激活中的作用，我们使用半胱氨酸诱变方法，其中S4片段的单个和多个精氨酸（R）残基的正电荷被中性电荷的氨基酸半胱氨酸（C）取代。为了确定是精氨酸残基本身还是其正电荷参与通道活化，构建了精氨酸至赖氨酸（R至K）突变。在tsA 201细胞中表达野生型（WT）和突变型NaChBac通道，并使用膜片钳技术的全细胞配置记录Na+电流。评价电流/电压（I-V）和电导/电压（G-P）关系、稳态失活（h（无穷大））和失活恢复，以确定S4突变对NaChBac通道生物物理特性的影响。S4片段上的R至C导致活化和失活动力学的减慢。精氨酸残基的电荷中和主要导致G-V和h（无穷大）曲线向更正的电位移动。G-V曲线移动与斜率降低相关，这可能反映了通道激活中涉及的门控电荷的减少。R114、R117或R120被C单次中和导致失活恢复非常缓慢。R111和R129的双重中和证实了R111在激活中的作用，并表明R129很可能不是电压传感器的一部分。大多数的R到K突变体保留WT样电流动力学，但表现出中间G-V曲线，稳态失活转移到更超极化的电位，和中间失活恢复的时间常数。这表明R在几个位置上在通道激活中起重要作用。这些数据是一致的概念，即S4是最有可能的NaChBac通道的电压传感器和正电荷和精氨酸残基的性质是必不可少的通道激活。
The one-domain voltage-gated sodium channel of Bacillus halodurans (NaChBac) is composed of six transmembrane segments (S1-S6) comprising a pore-forming region flanked by segments S5 and S6 and a voltage-sensing element composed of segment S4. To investigate the role of the S4 segment in NaChBac channel activation, we used the cysteine mutagenesis approach where the positive charges of single and multiple arginine (R) residues of the S4 segment were replaced by the neutrally charged amino acid cysteine (C). To determine whether it was the arginine residue itself or its positive charge that was involved in channel activation, arginine to lysine (R to K) mutations were constructed. Wild-type (WT) and mutant NaChBac channels were expressed in tsA201 cells and Na+ currents were recorded using the whole-cell configuration of the patch-clamp technique. The current/voltage (I-V) and conductance/voltage (G-P) relationships steady-state inactivation (h(infinity)) and recovery from inactivation were evaluated to determine the effects of the S4 mutations on the biophysical properties of the NaChBac channel. R to C on the S4 segment resulted in a slowing of both activation and inactivation kinetics. Charge neutralization of arginine residues mostly resulted in a shift toward more positive potentials of G-V and h(infinity) curves. The G-V curve shifts were associated with a decrease in slope, which may reflect a decrease in the gating charge involved in channel activation. Single neutralization of R114, R117, or R120 by C resulted in a very slow recovery from inactivation. Double neutralization of R111 and R129 confirmed the role of R111 in activation and suggested that R129 is most probably not part of the voltage sensor. Most of the R to K mutants retained WT-like current kinetics but exhibited an intermediate G-V curve, a steady-state inactivation shifted to more hyperpolarized potentials, and intermediate time constants of recovery from inactivation. This indicates that R, at several positions, plays an important role in channel activation. The data are consistent with the notion that the S4 is most probably the voltage sensor of the NaChBac channel and that both positive charges and the nature of the arginine residues are essential for channel activation.