The Distinct Roles of Calmodulin and Calmodulin Kinase II in the Reversal of Run-Down of L-Type Ca2+ Channels in Guinea-Pig Ventricular Myocytes
The Distinct Roles of Calmodulin and Calmodulin Kinase II in the Reversal of Run-Down of L-Type Ca2+ Channels in Guinea-Pig Ventricular Myocytes
复制标题
钙调蛋白和钙调蛋白激酶 II 在逆转豚鼠心室肌细胞 L 型 Ca2 通道衰退中的独特作用
DOI：
10.1254/jphs.09094fp
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发表时间：
2009-12-01
影响因子：
3.5
通讯作者：
Kameyama, Masaki
中科院分区：
文献类型：
--
作者：
Hao, Li-Ying;Wang, Wu-Yang;Kameyama, Masaki
在这项研究中，我们研究了钙调素激酶II（CaMK II）和钙调素（CaM）在逆转L-型钙通道下降中的作用。用膜片钳技术记录豚鼠心室肌细胞单个钙通道的活动，并在碱性内溶液中通过由内而外的膜片形成引起通道活动的下降。在1分钟后的补丁切除，1-30 μ M的CaMKII突变体T286 D（CaMKIIT 286 D），CaMKII的组成型活性类型，诱导的Ca 2+通道活性只有2%-10%的记录在细胞附着模式。然而，在CaMKIIT 286 D的存在下，钙调素的作用在逆转的下降的时间依赖性衰减被取消。制备了含有豚鼠Cav1.2（CT 1）C-末端区域的氨基酸1509-1789的GST融合蛋白。在下拉试验中，与用磷酸酶处理的CT 1相比，用CaMKIIT 286 D处理的CT 1显示出对CaM的更高亲和力。我们提出了一个模型，其中CaMKII介导的通道磷酸化调节CaM与通道的结合，以逆转L型Ca 2+通道的衰竭。
In this study, we investigated the roles of calmodulin kinase II (CaMKII) and calmodulin (CaM) in the reversal of run-down of L-type Ca2+ channels. Single Ca2+-channel activities in guinea-pig ventricular myocytes were recorded using the patch-clamp technique, and run-down of the channel activities was induced by inside-out patch formation in the basic internal solution. At 1 min after patch excision, 1-30 mu M CaMKII mutant T286D (CaMKIIT286D), a constitutively active type of CaMKII, induced the Ca2+-channel activities to only 2%-10% of that recorded in the cell-attached mode. However, in the presence of CaMKIIT286D, the time-dependent attenuation of CaM's effects in the reversal of run-down was abolished. A GST-fusion protein containing amino acids 1509-1789 of the C-terminal region of guinea-pig Cav1.2 (CT1) was prepared. In pull-down assays, CT1 treated with CaMKIIT286D showed a higher affinity for CaM compared with CT1 treated with phosphatase. We propose a model in which CaMKII-mediated phosphorylation of the channels regulates the binding of CaM to the channels in the reversal of run-down of L-type Ca2+ channels.