Accessibility of four arginine residues on the s4 segment of the Bacillus halodurans sodium channel
Accessibility of four arginine residues on the s4 segment of the Bacillus halodurans sodium channel
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DOI：
10.1007/s00232-007-9016-1
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发表时间：
2007-02-01
影响因子：
2.4
通讯作者：
Chahine, Mohamed
中科院分区：
文献类型：
--
作者：
Blanchet, Jonathan;Chahine, Mohamed
盐生芽孢杆菌的电压门控钠离子通道(NaChBac)由六个跨膜片段(S1-S6)组成，其中成孔区由片段S5和S6组成，电压敏感区由片段S1-S4组成。S4段构成了电压传感器的核心。我们研究了NaChBac S4片段上四个精氨酸残基的可及性，它们相互之间每隔三分之一的位置。通过定点突变，S4片段上的这些精氨酸残基被半胱氨酸取代。采用膜片钳全细胞记录技术记录Na+电流。我们测试了从细胞内和细胞外施加的巯基试剂在开放和闭合构象中的效果。基于最近结晶的KvAP和Kv1.2 K+通道，构建了NaChBac电压传感器的结构模型，以可视化精氨酸残基的可及性。我们的结果表明，精氨酸可及性在开放构象和闭合构象之间没有显著变化，支持S4片段在门控过程中小幅移动的想法。闭合构象的分子模拟也支持S4的小运动，其主要特征是沿孔的外围旋转和倾斜。有趣的是，S4片段的第二个精氨酸残基(R114)可以从闭合构象中的膜两侧的巯基试剂获得，根据我们的模型，似乎位于细胞内和细胞外水缝的交界处。
The voltage- gated Na+ channel of Bacillus halodurans ( NaChBac) is composed of six transmembrane segments ( S1 - S6), with a pore- forming region composed of segments S5 and S6 and a voltage- sensing domain composed of segments S1 - S4. The S4 segment forms the core of the voltage sensor. We explored the accessibility of four arginine residues on the S4 segment of NaChBac, which are positioned at every third position from each other. These arginine residues on the S4 segment were replaced with cysteines using site- directed mutagenesis. Na+ currents were recorded using the whole- cell configuration of the patch- clamp technique. We tested the effect of the sulfhydryl reagents applied from inside and outside the cellular space in the open and closed conformations. Structural models of the voltage sensor of NaChBac were constructed based on the recently crystallized KvAP and Kv1.2 K+ channels to visualize arginine residue accessibility. Our results suggest that arginine accessibility did not change significantly between the open and closed conformations, supporting the idea of a small movement of the S4 segment during gating. Molecular modeling of the closed conformation also supported a small movement of S4, which is mainly characterized by a rotation and a tilt along the periphery of the pore. Interestingly, the second arginine residue of the S4 segment ( R114) was accessible to sulfhydryl reagents from both sides of the membrane in the closed conformation and, based on our model, seemed to be at the junction of the intracellular and extracellular water crevices.