Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel -: Identification of the phosphorylation sites
Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel -: Identification of the phosphorylation sites
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DOI：
10.1074/jbc.m508661200
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发表时间：
2006-09-01
影响因子：
4.8
通讯作者：
Welling, Andrea
中科院分区：
文献类型：
--
作者：
Lee, Tae-Seong;Karl, Rosi;Welling, Andrea
L型钙通道的钙依赖性易化已被报道依赖于钙调蛋白激酶II的功能。相反，电压依赖性易化的机制尚不清楚。在表达Ca（v）1.2、Ca-v β 2a和钙调蛋白激酶II的HEK 293细胞中，在+30 mV下测量的钙电流被200 ms长的前脉冲促进高达1.5倍至+160 mV。这种电压依赖性的促进被钙调蛋白激酶II抑制剂KN 93和autocamtide-2相关肽阻止。在表达Cav1.2突变I1649 E的细胞中，Ca 2+结合钙调蛋白的结合的关键残基，促进作用也被废除。将钙调蛋白激酶II与来自鼠心脏和表达Cav1.2和钙调蛋白激酶II的HEK 293细胞的Cav1.2通道共免疫沉淀。在钙调蛋白和Ca ~（2+）存在下，沉淀的Ca（v）1.2通道被磷酸化。15个推定的钙调蛋白激酶II磷酸化位点主要在Ca（v）1.2的羧基末端尾中被鉴定。无论是在氨基酸1728截短，也不改变II-III环丝氨酸808和888丙氨酸影响促进钙电流。相反，单突变S1512 A和S1570 A降低易化，双突变S1512 A/S1570 A消除易化。这些丝氨酸侧接羧基末端EF-手基序。钙调蛋白激酶II与Ca（v）1.2通道的免疫沉淀不受突变S1512 A/S1570 A的影响。在S1512 A/S1570 A双突变体中，Ca（v）1.2蛋白的磷酸化强烈降低。这些结果表明，电压依赖性的Ca（v）1.2通道的易化依赖于钙调蛋白激酶II对Ser 1512/Ser 1570的磷酸化。
Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Ca(v)1.2, Ca-v beta 2a, and calmodulin kinase II, the calcium current measured at +30mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160mV. This voltage dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Cav1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Cav1.2 channel from murine heart and HEK 293 cells expressing Cav1.2 and calmodulin kinase II. The precipitated Ca(v)1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Ca(v)1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/ S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Ca(v)1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Ca(v)1.2 protein was strongly decreased in the S1512A/ S1570A double mutant. These results suggest that voltage dependent facilitation of the Ca(v)1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II.