A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating
A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating
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DOI：
10.1152/ajprenal.1997.273.4.f516
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发表时间：
1997-10-01
影响因子：
4.2
通讯作者：
Sackin, H
中科院分区：
文献类型：
--
作者：
Choe, H;Zhou, H;Sackin, H
ROMK通道通过控制哺乳动物皮层集合小管顶膜的钾分泌，在整体钾平衡中起关键作用。相反，家庭的强内向整流器（IRKs），ROMK通道是显着敏感的细胞内pH值。使用非洲爪蟾卵母细胞，我们已经证实了这种pH值的敏感性在单通道和全细胞水平。将卵母细胞pH从6.8降低至6.4（使用渗透性乙酸盐缓冲液）将通道开放概率从0.76 +/-0.02降低至接近零（n = 8），而不改变单通道电导。这是由于在低的内部pH下出现长寿命的封闭状态。我们已经证实，位于第一个假定的跨膜区段（hll）的NH 2末端的赖氨酸残基（ROMK 2上的K61; ROMK 1上的K80）主要负责赋予ROMK陡峭的pH敏感性（B. Fakler，J. Schultz，J. Yang，U.舒尔特大学Braandle，H.曾纳湖Y. Jan，and J. P. Ruppersberg. 15：4093-4099，1996）。然而，ROMK的表观pH值还取决于另一个高度保守的轻度疏水区域：ROMK 2上的T51（ROMK 1上的T70）。用带负电荷的谷氨酸替代该中性苏氨酸（T51），使内向电导的表观pK（a）从6.5 +/- 0.01（n = 8，野生型）变为7.0 +/- 0.02（n = 5，T51 E）。另一方面，用带正电荷的赖氨酸替换T51使表观pK（a）向相反方向移动，从6.5 +/- 0.01（n = 8，野生型）移动到6.0 +/- 0.02（n = 9，T51 K）。在位置51（ROMK 2）处的谷氨酸和赖氨酸取代的相反效应与其中T51在物理上接近K61并且通过静电机制改变局部pH或表观pH的模型一致。除了对pH敏感性的影响外，突变T51 E还将单通道电导从34.0 +/- 1.0 pS（n = 8，野生型）降低至17.4 +/- 1 pS（n = 9，T51 E），逆转了通道的电压门控，并显著增加了开放通道噪声。这些对单通道电流的影响表明，T51残基，位于ROMK 2的轻度疏水区域，也与渗透途径的疏水区域相互作用。
ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 +/- 0.02 to near zero (n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative transmembrane segment(hll), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Braandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093-4099, 1996). However, the apparent ph, of ROMK also depends on another residue (n a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent pK(a) for inward conductance from 6.5 +/- 0.01 (n = 8, wild type) to 7.0 +/- 0.02 (n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent pK(a) in the opposite direction, from 6.5 +/- 0.01 (n = 8, wild type) to 6.0 +/- 0.02 in = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent ph, via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 +/- 1.0 pS (n = 8, wild type) to 17.4 +/- 1 pS (n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.