Jiang L H, Gawler D J, Hodson N, Milligan C J, Pearson H A, Porter V, Wray D
School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
J Biol Chem. 2000 Mar 3;275(9):6135-43. doi: 10.1074/jbc.275.9.6135.
We have studied the effect of 8-bromo-cyclic GMP (8-Br-cGMP) on cloned cardiac L-type calcium channel currents to determine the site and mechanism of action underlying the functional effect. Rabbit cardiac alpha(1C) subunit, in the presence or absence of beta(1) subunit (rabbit skeletal muscle) or beta(2) subunit (rat cardiac/brain), was expressed in Xenopus oocytes, and two-electrode voltage-clamp recordings were made 2 or 3 days later. Application of 8-Br-cGMP caused decreases in calcium channel currents in cells expressing the alpha(1C) subunit, whether or not a beta subunit was co-expressed. No inhibition of currents by 8-Br-cGMP was observed in the presence of the protein kinase G inhibitor KT5823. Substitutions of serine residues by alanine were made at residues Ser(533) and Ser(1371) on the alpha(1C) subunit. As for wild type, the mutant S1371A exhibited inhibition of calcium channel currents by 8-Br-cGMP, whereas no effect of 8-Br-cGMP was observed for mutant S533A. Inhibition of calcium currents by 8-Br-cGMP was also observed in the additional presence of the alpha(2)delta subunit for wild type channels but not for the mutant S533A. These results indicate that cGMP causes inhibition of L-type calcium channel currents by phosphorylation of the alpha(1C) subunit at position Ser(533) via the action of protein kinase G.
我们研究了8-溴环鸟苷酸（8-Br-cGMP）对克隆的心脏L型钙通道电流的影响，以确定其功能效应背后的作用位点和机制。兔心脏α(1C)亚基，在有或无β(1)亚基（兔骨骼肌）或β(2)亚基（大鼠心脏/脑）存在的情况下，在非洲爪蟾卵母细胞中表达，2或3天后进行双电极电压钳记录。应用8-Br-cGMP导致表达α(1C)亚基的细胞中钙通道电流降低，无论是否共表达β亚基。在存在蛋白激酶G抑制剂KT5823的情况下，未观察到8-Br-cGMP对电流的抑制作用。在α(1C)亚基的Ser(533)和Ser(1371)残基处用丙氨酸取代丝氨酸残基。与野生型一样，突变体S1371A表现出8-Br-cGMP对钙通道电流的抑制作用，而突变体S533A未观察到8-Br-cGMP的作用。对于野生型通道，在额外存在α(2)δ亚基时也观察到8-Br-cGMP对钙电流的抑制作用，但突变体S533A则未观察到。这些结果表明，cGMP通过蛋白激酶G的作用，使α(1C)亚基在Ser(533)位点磷酸化，从而导致L型钙通道电流受到抑制。
