Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel
Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl- channel
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DOI：
10.1152/ajpcell.1998.275.4.c1113
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发表时间：
1998-10-01
影响因子：
5.5
通讯作者：
Cuppoletti, J
中科院分区：
文献类型：
--
作者：
Stroffekova, K;Kupert, EY;Cuppoletti, J
兔和人的ClC-2G Cl-通道是电压敏感的，并被蛋白激酶A和低细胞外ph激活。本研究的目的是研究ClC-2G Cl-通道酸激活的机制，并确定哪些氨基酸残基在酸激活中起作用。在+/-80 mV保持电位下，通道打开概率（P-o）随细胞外H+浓度（即细胞外pH， pH（反式））呈浓度依赖性增加4倍，明显的酸性解离常数为pH 4.95 +/- 0.27。在pH（反式）7.4时，1-乙基-3（3-二甲氨基丙基）碳二酰亚胺催化的甘氨酸甲酯酰胺化使通道的P-o增加了3倍，而通道通常表现为低P-o。随着细胞外pH值的降低（质子化）或酰胺化，P-o的增加是由于通道门控的打开时间常数的显著增加和关闭时间常数的显著减少，这种影响对施加电压不敏感。利用定点诱变技术，确定细胞外区域EELE（氨基酸416-419）为pH传感器，并发现氨基酸Glu-419在细胞外酸激活ClC-2G Cl-通道中起关键或主导作用。
Rabbit and human ClC-2G Cl- channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl- channel and to determine which amino acid residues play a role in this acid activation. Channel open probability (P-o) at +/-80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+ concentration (that is, extracellular pH, pH(trans)), with an apparent acidic dissociation constant of pH 4.95 +/- 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased P-o threefold at pH(trans) 7.4, at which the channel normally exhibits low P-o. With extracellular pH reduction (protonation) or amidation, increased P-o was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416-419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl- channel by extracellular acid.