Enhanced Activity of a Large Conductance, Calcium-sensitive K+ Channel in the Presence of Src Tyrosine Kinase

Abstract

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.In the large family of voltage-gated K ϩ channels, large conductance, calcium-sensitive potassium (maxi-K or BK Ca ) 1 channels represent a unique class whose gating depends primarily on membrane voltage but which can be shifted in the negative direction by intracellular free calcium. A direct physiologic consequence of this behavior is that BK Ca channels act as "coincidence detectors" and regulate, in a feedback fashion, cellular processes stimulated by close temporal changes in membrane potential and intracellular calcium. That BK Ca channels indeed play such a role is evidenced by the fact that blocking these channels increases the degree of myogenic tone observed in arterial smooth muscle (1-3) and enhances the presynaptic calcium-dependent release of neurotransmitter at neuromuscular junctions (4, 5).Given their potential to influence cellular processes, it is not surprising that BK Ca channels are also targets of cellular signaling pathways, including phosphorylation and dephosphorylation reactions (6 -11), heterotrimeric GTP-binding proteins (12, 13), and the endothelium-derived vasodilator nitric oxide (14). To date, however, many of the molecular aspects of these regulatory events remain poorly understood.Of these various cellular pathways, protein phosphorylation remains as one of the most common forms of intrace...

Leucine Zipper Domain Targets cAMP-dependent Protein Kinase to Mammalian BK Channels

“…In these assays we stimulated PKA activity intimately associated with the channel by applying cAMP to the intracellular face of isolated inside-out patches. As previously reported (17) the effects of cAMP in this system are dependent upon the presence of Mg-ATP, and the actions of cAMP are completely abolished by the PKA inhibitor peptide PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] . Although STREX channels are inhibited whereas ZERO channels are activated by PKA closely associated with the channel (17), the short LZ1-competing peptides effectively blocked PKAdependent regulation of either splice variant.…”

“…Phenotypic variation in native BK channels results from extensive alternative exon splicing of the ␣-subunit (6, 9, 10) as well as through interaction with regulatory ␤-subunits and accessory proteins (11)(12)(13). The BK channel ␣-subunit is a target for regulation by multiple protein kinases and protein phosphatases (3, 14 -18), and several protein kinases have been reported to co-immunoprecipitate with mammalian BK channels (3,16,17). Although several consensus phosphorylation sites have been identified by mutagenesis within the intracellular C-terminal domain, BK channel ␣-subunits do not contain previously identified protein-protein interaction domains.…”

α5β1 Integrin Engagement Increases Large Conductance, Ca2+-activated K+ Channel Current and Ca2+ Sensitivity through c-src-mediated Channel Phosphorylation

“…We have previously shown that activation of VSM ␣5␤1 integrins leads to potentiation of L-type calcium channel current through the involvement of the integrin-associated tyrosine kinases c-src and focal adhesion kinase (13,14). Other studies have reported that BK channel activity is enhanced following direct channel phosphorylation by other src family kinases and by Pyk2 (proline-rich tyrosine kinase 2) (7,8). Recently, we found that the endogenous ␣5␤1 integrin ligand fibronectin (FN) potentiates BK channel current in VSM cells (15).…”

“…Phosphorylation of the BK channel by tyrosine kinases, including focal adhesion kinase (6) and c-src (7,8), suggests a possible involvement of the channel in integrin signaling. Integrins are a widely expressed class of adhesion receptor whose interactions with extracellular matrix (ECM) proteins lead to activation of signaling cascades, including protein tyrosine phosphorylation (9).…”

Association of β-catenin with the α-subunit of neuronal large-conductance Ca 2+ -activated K+ channels

“…In Drosophila, two cytosolic regulatory proteins, Slob and dSLIP, and two protein kinases, Src and PKAc, are able to assemble with the slo ␣-subunit (27)(28)(29). In vertebrates, however, only kinases, such as Src and Syk, have been reported to interact with this subunit (30)(31)(32). To seek other proteins that associate with K Ca channels and mediate their presynaptic targeting, we used yeast two-hybrid screening to search a cDNA library made from sensory epithelia of the chicken's cochlea.…”