Involvement of histidine residues in proton sensing of ROMK1 channel
Involvement of histidine residues in proton sensing of ROMK1 channel
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DOI：
10.1074/jbc.275.11.7811
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发表时间：
2000-03-17
影响因子：
4.8
通讯作者：
Jiang, C
中科院分区：
文献类型：
--
作者：
Chanchevalap, S;Yang, ZJ;Jiang, C
ROMK通道受到细胞内酸化的抑制，这种pH敏感性与通道蛋白中的几个氨基酸残基如Lys-61、Thr-51和His-206有关与所有其他氨基酸不同，组氨酸在pH 6-7下可滴定，在pH 6以下携带正电荷。为了测试某些组氨酸残基参与ROMK 1的CO2和pH传感的假设，我们用定点突变的方法对通道中的所有组氨酸残基进行了系统突变实验，在N端有两个组氨酸残基。His-23、His-31或两者的突变不影响通道对CO2的敏感性。6个组氨酸残基位于C末端。His-225、His-274、His-342和His-354在CO2和pH传感中是关键的。它们中的任一个的突变分别使CO2和pH敏感性降低20-50%和类似于0.2个pH单位。所有这些基因的同时突变消除了CO2敏感性，并导致该突变通道仅对极酸性pH作出反应，His-280的类似突变没有影响。His-270在CO2和pH传感中的作用尚不清楚，因为用中性、负性或正性氨基酸取代该残基不会产生任何功能通道。因此，这些结果表明组氨酸残基有助于ROMK 1通道对高碳酸血症和细胞内酸中毒的敏感性。
ROMK channels are inhibited by intracellular acidification, This pH sensitivity is related to several amino acid residues in the channel proteins such as Lys-61, Thr-51, and His-206 (in ROMK2), Unlike all other amino acids, histidine is titratable at pH 6-7 carrying a positive charge below pH 6, To test the hypothesis that certain histidine residues are engaged in CO2 and pH sensing of ROMK1, we performed experiments by systematic mutations of all histidine residues in the channel using the site-directed mutagenesis, There are two histidine residues in the N terminus. Mutations of His-23, His-31, or both together did not affect channel sensitivity to CO2. Six histidine residues are located in the C terminus. His-225, His-274, His-342, and His-354 were critical in CO2 and pH sensing. Mutation of either of them reduced CO2 and pH sensitivities by 20-50% and similar to 0.2 pH units, respectively. Simultaneous mutations of all of them eliminated the CO2 sensitivity and caused this mutant channel to respond to only extremely acidic pH, Similar mutations of His-280 had no effect. The role of His-270 in CO2 and pH sensing is unclear, because substitutions of this residue with either a neutral, negative, or positive amino acid did not produce any fumctional channel. These results therefore indicate that histidine residues contribute to the sensitivity of the ROMK1 channel to hypercapnia and intracellular acidosis.