Enhanced activity of a large conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase

Large conductance, calcium-sensitive K+ channels (BKCa channels) contribute to the control of membrane potential in a variety of tissues, including sm ooth muscle, where they act as the target effector for intracellular "calci um sparks" and the endothelium-derived vasodilator nitric oxide. Various si gnal transduction pathways, including protein phosphorylation can regulate the activity of BKCa channels, along with many other membrane ion channels. In our study, we have examined the regulation of BKCa channels by the cell ular Src gene product (cSrc), a soluble tyrosine kinase that has been impli cated in the regulation of both voltage and ligand-gated ion channels. Usin g a heterologous expression system, we observed that co-expression of murin e BKCa channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BKCa, channel activity in excised membran e patches. In contrast, co-expression with a catalytically inactive cSrc mu tant produced no change in BKCa, channel activity, demonstrating the requir ement for a functional cSrc molecule. Furthermore, we observed that BKCa ch annels underwent direct tyrosine phosphorylation in cells co-transfected wi th BKCa, channels and active cSre 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 phosphoryla tion, Given that cSre may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyr osine phosphorylation of BKCa, channels in situ may represent a novel regul atory mechanism for altering membrane potential and calcium entry.