From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
J Biol Chem. 2010 Feb 5;285(6):3777-3783. doi: 10.1074/jbc.M109.084368. Epub 2009 Dec 3.
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 approximately 1 at pH 4 and approximately 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.
胞质 pH 从 7 变为 4 会打开细菌钾通道 KcsA。然而，导致通道打开的整体门控机制，特别是细胞质结构域的贡献，仍未解决。在这里，我们报告说，删除细胞质结构域会导致通道在 pH 4 时的电导和门控行为发生变化，而在 pH 7 时则不会打开通道。为了探测通道打开过程中细胞质结构域的重排，用半胱氨酸取代了氨基酸残基，并标记了一种荧光团（四甲基罗丹明马来酰亚胺），该荧光团在从亲水环境转移到疏水环境时会增加荧光强度。在所有情况下，通道开放概率（P(o)）在 pH 4 时约为 1，在 pH 7 时约为 0。当 pH 从 7 变为 4 时，在细胞质结构域中用四甲基罗丹明马来酰亚胺标记的残基的荧光强度有明显增加，这表明荧光团从亲水环境转移到了疏水环境。二吡咯甲酰胺，一种脂溶性淬灭剂，降低了 pH 4 时细胞质结构域中标记残基的荧光强度。这些结果表明，pH 的降低会导致 KcsA 细胞质结构域中与通道打开相关的主要构象重排。
