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

点击翻译按钮获取中文摘要

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.