Ling S, Woronuk G, Sy L, Lev S, Braun A P
Smooth Muscle Research Group, Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
J Biol Chem. 2000 Sep 29;275(39):30683-9. doi: 10.1074/jbc.M004292200.
Large conductance, calcium-sensitive K(+) channels (BK(Ca) channels) contribute to the control of membrane potential in a variety of tissues, including smooth muscle, where they act as the target effector for intracellular "calcium sparks" and the endothelium-derived vasodilator nitric oxide. Various signal transduction pathways, including protein phosphorylation can regulate the activity of BK(Ca) channels, along with many other membrane ion channels. In our study, we have examined the regulation of BK(Ca) channels by the cellular Src gene product (cSrc), a soluble tyrosine kinase that has been implicated in the regulation of both voltage- and ligand-gated ion channels. Using a heterologous expression system, we observed that co-expression of murine BK(Ca) channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BK(Ca) channel activity in excised membrane patches. In contrast, co-expression with a catalytically inactive cSrc mutant produced no change in BK(Ca) channel activity, demonstrating the requirement for a functional cSrc molecule. Furthermore, we observed that BK(Ca) channels underwent direct tyrosine phosphorylation in cells co-transfected with BK(Ca) channels and active cSrc but not in cells co-transfected with the kinase inactive form of the enzyme. A single Tyr to Phe substitution in the C-terminal half of the channel largely prevented this observed phosphorylation. Given that cSrc may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyrosine phosphorylation of BK(Ca) channels in situ may represent a novel regulatory mechanism for altering membrane potential and calcium entry.
大电导钙敏感钾通道（BK（Ca）通道）有助于控制多种组织中的膜电位，包括平滑肌，在平滑肌中它们作为细胞内“钙火花”和内皮源性血管舒张剂一氧化氮的靶效应器。包括蛋白质磷酸化在内的各种信号转导途径可以调节BK（Ca）通道以及许多其他膜离子通道的活性。在我们的研究中，我们研究了细胞Src基因产物（cSrc）对BK（Ca）通道的调节作用，cSrc是一种可溶性酪氨酸激酶，与电压门控离子通道和配体门控离子通道的调节均有关联。使用异源表达系统，我们观察到在HEK 293细胞中鼠BK（Ca）通道与人cSrc酪氨酸激酶的共表达导致在切除的膜片中BK（Ca）通道活性出现钙敏感增强。相反，与催化失活的cSrc突变体共表达不会使BK（Ca）通道活性发生变化，这表明需要功能性的cSrc分子。此外，我们观察到在与BK（Ca）通道和活性cSrc共转染的细胞中BK（Ca）通道发生直接酪氨酸磷酸化，但在与该酶的激酶失活形式共转染的细胞中未发生。通道C末端一半的单个酪氨酸突变为苯丙氨酸很大程度上阻止了观察到的这种磷酸化。鉴于cSrc可能被受体酪氨酸激酶或G蛋白偶联受体激活，这些发现表明原位BK（Ca）通道的cSrc依赖性酪氨酸磷酸化可能代表一种改变膜电位和钙内流的新型调节机制。
