Regulation of Kir channels by intracellular pH and extracellular K(+): mechanisms of coupling.
Regulation of Kir channels by intracellular pH and extracellular K(+): mechanisms of coupling.
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DOI：
10.1085/jgp.200308989
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发表时间：
2004-04
期刊：
影响因子：
--
通讯作者：
Palmer LG
中科院分区：
文献类型：
--
作者：
Dahlmann A;Li M;Gao Z;McGarrigle D;Sackin H;Palmer LG
ROMK通道受细胞内pH(Phi)和细胞外K+(K+o)的调节。在非洲爪哇卵母细胞中表达后，研究了这种调节的机制。用pH不敏感通道IRK1(KIR2.1)的相应区域替换ROMK2(Kir1.1b)的COOH末端部分，产生了一个嵌合通道(称为C13)，该通道对细胞内H+的抑制具有增强的敏感性，使抑制的表观pKa增加了∼0.9pH单位。第二跨膜螺旋COOH末端的三个氨基酸取代(I159V、L160M和I163M)解释了这些影响。阳离子激活通道的离子选择性顺序为K+≅Rb+&Gt；NH4+&Gt；&Gt；Na+，与离子通透性类似，提示与选择性过滤器的相互作用。我们测试了一个耦合模型，在这个模型中，一个pH敏感的门可以从内部关闭气孔，防止K+从细胞质进入，并增加选择性过滤器对K+o去除的敏感度。我们利用正膜电位模拟关闭这个门，以引起细胞内阳离子的阻断。当K+O在10到110 mm之间时，这会导致缓慢的可逆的电导下降。然而，附加的通道结构，其中保持了内向整流，但取消了pH传感器，在相同的条件下不能对电压做出反应。这表明，阻止细胞内K+对选择性过滤器的访问不能解释耦合。C13嵌合体对胞外Ba2+阻断的敏感性是ROMK2的10倍，表明COOH末端的变化影响了离子与孔外部分的结合。这种效应与其对H+失活的敏感性有关。我们的结论是，通过改变选择性滤光片的性质，降低phi增加了ROMK2通道对K+o的敏感性。
ROMK channels are regulated by internal pH (pHi) and extracellular K+ (K+ o). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H+, increasing the apparent pKa for inhibition by ∼0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K+ o, consistent with coupling between the responses to pHi and K+ o. The ion selectivity sequence of the activation of the channel by cations was K+ ≅ Rb+ > NH4 + >> Na+, similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K+ from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K+ o. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K+ o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K+ to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba2+ block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H+. We conclude that decreasing pHI increases the sensitivity of ROMK2 channels to K+ o by altering the properties of the selectivity filter.
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影响因子：
56.9
作者：
Doyle, DA;Cabral, JM;MacKinnon, R
通讯作者：
MacKinnon, R
影响因子：
3.8
作者：
Holmgren, M;Smith, PL;Yellen, G
通讯作者：
Yellen, G
影响因子：
3.8
作者：
Choe, H;Sackin, H;Palmer, LG
通讯作者：
Palmer, LG
影响因子：
4.8
作者：
Chanchevalap, S;Yang, ZJ;Jiang, C
通讯作者：
Jiang, C
DOI：
10.1085/jgp.58.4.413
发表时间：
1971-10
期刊：
The Journal of general physiology
影响因子：
--
作者：
Armstrong CM
通讯作者：
Armstrong CM