Abstract
Large conductance calcium- and voltage-activated potassium (BK) channels assemble as macromolecular signaling complexes and are potently regulated by reversible protein phosphorylation. However, although numerous studies have revealed regulation of BK channels through changes in direct phosphorylation of the pore-forming alpha-subunits the functional role of changes in phosphorylation of defined adapter/signaling proteins within the complex on channel function are essentially not known. Here, we demonstrate that mammalian BK channels are potently regulated by endogenous protein-tyrosine kinase and protein-tyrosine phosphatase activity closely associated with the channel. BK channel regulation was not dependent upon direct phosphorylation of the BK alpha-subunit, rather channel function was controlled by the tyrosine phosphorylation status of the adapter protein cortactin that assembles directly with the BK channel. Our data thus reveal a novel mode for BK channel regulation by reversible tyrosine phosphorylation and strongly support the hypothesis that phosphorylation-dependent regulation of accessory proteins within the BK channel signaling complex represents an important target for control of BK channel function.
Highlights
BK channels exist as a multimolecular signaling complex [8] suggesting that an alternative mechanism of BK channel regulation may be mediated through phosphorylation-dependent regulation of proteins closely associated with the BK channel pore-forming subunits
Several independent lines of evidence support direct tyrosine kinasemediated phosphorylation of the BK channel ␣-subunit, including: (i) overexpression of active c-Src, pyk2, and HCK tyrosine kinases results in phosphorylation of the ␣-subunits expressed in HEK cell lines [1, 11, 12]; (ii) site-directed mutagenesis of a C-terminal tyrosine residue (Tyr-762) abolishes channel phosphorylation and regulation by overexpressed c-Src [11]; (iii) prostaglandin E2 stimulation results in phosphorylation of BK channel ␣-subunits in osteocarcinoma cells [13]; (iv) overexpressed c-Src co-localizes with BK channels expressed in HEK293 cells [1] and endogenous c-Src co-immu
Associated Endogenous Tyrosine Kinase/Phosphatase Activity Regulates BK Channels—As a first step to interrogate the role of endogenous Src family tyrosine kinases, closely associated with BK channels, we selected inside-out patches from HEK293 cells in which BK channels were expressed at low levels to allow analysis at single channel resolution and to reduce the potential saturation of endogenous signaling cascades that may be inherent in highly overexpressing cells in which large macropatch recordings are routinely analyzed
Summary
HEK293 Cell Culture and Transfection—HEK293 cells, which do not express endogenous BK channels as determined by reverse transcription-PCR, Western blot, and functional analysis, were subcultured essentially as described [4, 21]. Patch Clamp Electrophysiology—Experiments were performed by only selecting patches that contained low channel densities To facilitate this when using stable cell lines, cells were not maintained in selection medium. The integral divided by integration time and single-channel current amplitude gives N*Po. To determine the mean percent (%) change in channel activity after a treatment, in patches with low to moderate levels of channel expression, mean Po or N*Po was measured immediately before and 10 min after the respective drug treatment. Cells were washed three times in ice-cold PBS, permeabilized using 0.1% Triton in PBS at room temperature for 10 min, washed three times, and blocked in PBS containing 1% bovine serum albumin and 0.05% Tween 20 for 1 h. Data were analyzed by analysis of variance (ANOVA) with posthoc Student-Newman-Keuls test with significance set at p Ͻ 0.01, or by Students paired t test, as appropriate
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