Rearrangements in the KcsA Cytoplasmic Domain Underlie Its Gating
Rearrangements in the KcsA Cytoplasmic Domain Underlie Its Gating
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DOI：
10.1074/jbc.m109.084368
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发表时间：
2010-02-05
影响因子：
4.8
通讯作者：
Ide, Toru
中科院分区：
文献类型：
--
作者：
Hirano, Minako;Takeuchi, Yuko;Ide, Toru
细胞质pH值从7变为4时，细菌钾通道KcsA打开。然而，导致通道开放的整体门控机制，特别是胞质结构域的贡献，仍然没有解决。在这里，我们报告，删除的胞质结构域导致在pH 4的通道电导和门控行为的变化，而在pH 7的通道开放。为了探测通道开放期间胞质结构域中的重排，将氨基酸残基用半胱氨酸取代并用荧光团（四甲基罗丹明马来酰亚胺）标记，所述荧光团在从亲水性环境转移到疏水性环境时表现出增加的荧光强度。在所有情况下，通道开放概率（P-o）在pH 4下类似于1，在pH 7下类似于0。当pH从7变为4时，观察到细胞质结构域中四甲基罗丹明马来酰亚胺标记的残基的荧光强度的主要增加，这表明荧光团从亲水性环境转移到疏水性环境。双苦酰胺，一种脂溶性淬灭剂，在pH 4时，降低了胞质结构域中标记残基的荧光强度。这些结果表明，在pH值的降低引入主要的构象重排与通道开放的KcsA胞质结构域。
A change of cytosolic pH 7 to 4 opens the bacterial potassium channel KcsA. However, the overall gating mechanism leading to channel opening, especially the contribution of the cytoplasmic domain, remains unsolved. Here we report that deletion of the cytoplasmic domain resulted in changes in channel conductance and gating behavior at pH 4 without channel opening at pH 7. To probe for rearrangements in the cytoplasmic domain during channel opening, amino acid residues were substituted with cysteines and labeled with a fluorophore (tetramethylrhodamine maleimide) that exhibits increased fluorescence intensity upon transfer from a hydrophilic to hydrophobic environment. In all cases channel open probability (P-o) was similar to 1 at pH 4 and similar to 0 at pH 7. Major increases in fluorescence intensity were observed for tetramethylrhodamine maleimide-labeled residues in the cytoplasmic domain as pH changed from 7 to 4, which suggests the fluorophores shifted from a hydrophilic to hydrophobic environment. Dipicrylamide, a lipid soluble quencher, reduced the fluorescence intensities of labeled residues in the cytosolic domain at pH 4. These results reveal that a decrease in pH introduces major conformational rearrangements associated with channel opening in the KcsA cytoplasmic domain.