Laboratory for Cell Dynamics Observation, Quantitative Biology Center, RIKEN, Osaka, Japan.
Biophys J. 2011 Nov 2;101(9):2157-62. doi: 10.1016/j.bpj.2011.09.024. Epub 2011 Nov 1.
The KcsA channel is a representative potassium channel that is activated by changes in pH. Previous studies suggested that the region that senses pH is entirely within its transmembrane segments. However, we recently revealed that the cytoplasmic domain also has an important role, because its conformation was observed to change dramatically in response to pH changes. Here, to investigate the effects of the cytoplasmic domain on pH-dependent gating, we made a chimera mutant channel consisting of the cytoplasmic domain of the KcsA channel and the transmembrane region of the MthK channel. The chimera showed a pH dependency similar to that of KcsA, indicating that the cytoplasmic domain can act as a pH sensor. To identify how this region detects pH, we substituted certain cytoplasmic domain amino acids that are normally negatively charged at pH 7 for neutral ones in the KcsA channels. These mutants opened independently of pH, suggesting that electrostatic charges have a major role in the cytoplasmic domain's ability to sense and respond to pH.
KcsA 通道是一种代表性的钾通道，其活性受 pH 值变化的影响。先前的研究表明，感知 pH 值的区域完全位于其跨膜片段内。然而，我们最近揭示，细胞质结构域也具有重要作用，因为观察到其构象在响应 pH 值变化时会发生剧烈变化。在这里，为了研究细胞质结构域对 pH 依赖性门控的影响，我们构建了一个嵌合突变体通道，由 KcsA 通道的细胞质结构域和 MthK 通道的跨膜区域组成。该嵌合体表现出与 KcsA 相似的 pH 值依赖性，表明细胞质结构域可以作为 pH 值传感器。为了确定该区域如何检测 pH 值，我们将 KcsA 通道中正常带负电荷的某些细胞质结构域氨基酸替换为 pH 值为 7 时的中性氨基酸。这些突变体独立于 pH 值而开放，表明静电电荷在细胞质结构域感知和响应 pH 值的能力中起着主要作用。
