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Mechanisms for regulation of L-type Ca channels by intracellular factors

细胞内因子调节L型Ca通道的机制

基本信息

批准号：
13670045
负责人：
KAMEYAMA Masaki
金额：
$ 2.18万
依托单位：
Kagoshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670045/
关键词：
Calcium channel ion channel patch clamp cardiac myocyte Caチャンネルイオンチャンネル

项目摘要

L-type Ca channels are present in many cell types and control various cellular functions. A unique feature of L-type Ca channels is 'run-down' phenomenon, which is prominent in cell-free patch-clamp experiments, in this study, we tested the hypothesis that 'run-down' might be caused by a loss of unknown regulatory factor(s) in cytoplasm. In addition, we also explored phosphorylation site(s) of the channel catalyzed by protein kinase A (PKA) and possible interactions between the actions mediated by cytoplasmic factors and PKA. Electrophysiological experiments were carried out in guinea-pig ventricular myocytes or in cultured BHK cells expressing guinea-pig Ca channels, using patch-clamp method. Results: 1)One factor appreared at Mr' of 250-300k on gel filtration, and suggested to be calpastatin. Another factor (H factor) appeared at Mr' of >300k on gel filtration, and was eluted by KCl of 180-360 mM. 2)Calpstatin interacted with a fragment of C-terminal region of the channel in surface prasmon resonance (SPR) analysis. 3)Substitution by alanin of Ser1574 or Sen1927, located in the C-terminal tail of the channel, affected the up-modulation of channel activity by PKA. These results suggest that L-type Ca channel is regulated by cytoplasmic factors and PKA, and that the C-terminal tail of the channel is involved in these regulatory mechanisms.

L-型Ca通道存在于许多细胞类型中并控制各种细胞功能。L-型钙通道的一个独特特征是"run-down"现象，这在无细胞膜片钳实验中是突出的，在本研究中，我们验证了"run-down"可能是由细胞质中未知调节因子的丢失引起的假设。此外，我们还探讨了蛋白激酶A（PKA）催化的通道的磷酸化位点以及胞质因子介导的作用与PKA之间可能的相互作用。采用膜片钳方法，在豚鼠心室肌细胞或表达豚鼠Ca通道的培养BHK细胞中进行电生理实验。结果：1）凝胶过滤在Mr '为250 - 300K时出现一个因子，推测为钙蛋白酶抑制蛋白。另一个因子（H因子）在凝胶过滤时出现在Mr '> 300k处，用180 - 360mM的KCl洗脱。2）表面共振（SPR）分析表明，Calpstatin与通道的C-末端区域的一个片段相互作用。3)丙氨酸取代Ser1574或Sen1927，位于通道的C-末端尾部，影响PKA的通道活性的上调。这些结果表明，L-型钙通道受细胞质因子和PKA的调节，通道的C-末端尾参与了这些调节机制。