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...

1. The large conductance, calcium-sensitive K + channel (BK Ca channel) is a unique member of the K + -selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltagedependent gating in a negative or left-ward direction, thereby adjusting channel opening to changes in cellular membrane potential.2. We hypothesized that the intrinsic calcium-binding site within the BK Ca channel a subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the _z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites.3. Using macroscopic current recordings of wild-type and mutant BK Ca channels in excised insideout membrane patches from HEK 293 cells, we observed that a single point mutation in the C-terminus (Site 6, FLD 923 QD to N), adjacent to the 'calcium bowl' described by Salkoff and colleagues, shifted calcium-sensitive gating right-ward by 50-65 mV over the range of 2-12 µM free calcium, but had little effect on voltage-dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 (PVD 81 EK to N) in the N-terminus produced a greater shift (70-90 mV) in calcium-sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity ~11-fold (129.5 µM vs. 11.3 µM) compared to the wild-type channel.4. We further observed that a bacterially expressed protein encompassing Site 6 of the BK Ca channel C-terminus and bovine brain calmodulin were both able to directly bind 45 Ca 2+ following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE).5. Our results suggest that two regions within the mammalian BK Ca channel a subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel.

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