Structural determinants and specificities for ROMK1-phosphoinositide interaction.
Structural determinants and specificities for ROMK1-phosphoinositide interaction.
复制标题
ROMK1-磷酸肌醇相互作用的结构决定因素和特异性。
DOI：
10.1152/ajprenal.00300.2001
复制
发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Huang,Chou-Long
中科院分区：
文献类型：
--
作者：
Zeng,Wei-Zhong;Liou,Horng-Huei;Krishna,UMurali;Falck,JR;Huang,Chou-Long
我们最近报道了磷脂酰肌醇二磷酸 (PIP2) 和 ROMK1 COOH 末端胞质结构域之间的直接相互作用对于通道的开放非常重要。我们确定 ROMK1 的精氨酸 188 是这种相互作用的关键残基。在这里，我们进一步报道，用中性氨基酸替代赖氨酸-181、精氨酸-217或赖氨酸-218会降低全电导状态的单通道打开概率，并增加亚电导状态的打开频率。与野生型ROMK1通道相比，这些取代突变体还表现出对抗PIP2抗体的阻断和细胞内质子的抑制的敏感性增加。这些结果表明，与精氨酸-188 一样，赖氨酸-181、精氨酸-217 和赖氨酸-218 也参与与 PIP2 的相互作用，并且对于 ROMK1 在完全电导下打开至关重要。使用在头基不同位置含有磷酸盐的合成磷酸肌醇，我们还检查了磷酸肌醇在ROMK1通道调节中的特异性。我们发现肌醇头基的4位和5位均含有磷酸盐的磷酸肌醇在激活ROMK1通道方面具有最高的功效。这些结果表明磷脂酰肌醇 4,5-二磷酸可能是调节生理膜环境中 ROMK1 通道的重要磷酸肌醇。
We have recently reported that direct interaction between phosphatidylinositol bisphosphate (PIP2) and the COOH-terminal cytoplasmic domain of ROMK1 is important for opening of the channel. We identified arginine-188 of ROMK1 as a critical residue for this interaction. Here, we further report that substitution of a neutral amino acid for lysine-181, arginine-217, or lysine-218 decreases single-channel open probability for the full-conductance state and increases the frequency of opening at a subconductance state. Compared with wild-type ROMK1 channels, these substitution mutants also display an increased sensitivity to the block by anti-PIP2antibodies and to inhibition by intracellular protons. These results indicate that, like arginine-188, lysine-181, arginine-217, and lysine-218 are also involved in interactions with PIP2and are critical for ROMK1 to open at full conductance. Using synthetic phosphoinositides containing phosphates at different positions in the head group, we also examined the specificities of phosphoinositides in the regulation of ROMK1 channels. We found that phosphoinositides containing phosphate at bothpositions 4and5of the inositol head group have the highest efficacy in activating ROMK1 channels. These results suggest that phosphatidylinositol 4,5-bisphosphate is likely the important phosphoinositide in the regulation of ROMK1 channels in a physiological membrane milieu.