Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.
Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.
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DOI：
10.1152/ajpcell.1995.268.1.c191
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
D. Malinowska;E. Kupert;A. Bahinski;A. M. Sherry;J. Cuppoletti
中科院分区：
文献类型：
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作者：
D. Malinowska;E. Kupert;A. Bahinski;A. M. Sherry;J. Cuppoletti
从兔胃文库中分离编码Cl-通道的cDNA，测序，并在非洲爪蟾卵母细胞中表达。预测的蛋白质（898个氨基酸，相对分子质量98，433 Da）与大鼠脑ClC-2 Cl-通道总体相似性为93%。然而，朝向COOH-末端的151个氨基酸延伸与缺失6个氨基酸的ClC-2相似性为74%。引入了两个新的潜在的蛋白激酶A（PKA）磷酸化位点（也是蛋白激酶C磷酸化位点）。注射cRNA的非洲爪蟾卵母细胞表达在pHtrans 3下有活性的Cl-通道，并且在800 mM CsCl下具有线性电流-电压（I-V）曲线和29 +/- 1 pS的斜率电导。五倍Cl-梯度引起I-V曲线的1/3位移，反转电位为+30 +/-3 mV，表明阴离子选择性。选择性为I- > Cl- > NO3-。天然和重组的Cl-通道在体外均被PKA催化亚基和ATP激活。克隆的和天然通道的电生理和调节特性是相似的。克隆的蛋白质可能是参与胃HCl分泌的Cl-通道。
cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.