Schulte U, Hahn H, Wiesinger H, Ruppersberg J P, Fakler B
Department of Physiology II, Ob dem Himmelreich 7, 72074 Tübingen, Germany.
J Biol Chem. 1998 Dec 18;273(51):34575-9. doi: 10.1074/jbc.273.51.34575.
ROMK channels (Kir1.1) are members of the superfamily of inward rectifier potassium channels (Kir) and represent the channels underlying K+ secretion in the kidney. As their native counterparts, Kir1.1 channels are gated by intracellular pH, with acidification leading to channel closure. Although a lysine residue (Lys80) close to the first hydrophobic segment M1 has been identified as the pH sensor, little is known about how opening and closing of the channel is accomplished. Here we investigate the gating process of Kir1.1 channels exploiting their state-dependent modification by water-soluble oxidants and sulfhydryl reagents. Mutagenesis of all intracellular cysteines either alone or in combination revealed two residues targeted by these reagents, one in the N terminus (Cys49) and one in the C terminus (Cys308) of the channel protein. Both sites reacted with the thiol reagents only in the closed state and not in the open state. These results indicate that pH-dependent gating of Kir1.1 channels involves movement of protein domains in both N and C termini of the Kir1.1 protein.
ROMK通道（Kir1.1）是内向整流钾通道（Kir）超家族的成员，代表肾脏中钾离子分泌的基础通道。与它们的天然对应物一样，Kir1.1通道受细胞内pH值门控，酸化会导致通道关闭。尽管已确定靠近第一个疏水片段M1的赖氨酸残基（Lys80）为pH传感器，但对于通道的开启和关闭过程如何完成知之甚少。在这里，我们利用水溶性氧化剂和巯基试剂对Kir1.1通道的状态依赖性修饰来研究其门控过程。单独或组合对所有细胞内半胱氨酸进行诱变，发现了这些试剂作用的两个残基，一个在通道蛋白的N端（Cys49），另一个在C端（Cys308）。这两个位点仅在关闭状态下与巯基试剂反应，而在开放状态下不反应。这些结果表明，Kir1.1通道的pH依赖性门控涉及Kir1.1蛋白N端和C端蛋白质结构域的移动。
