Large-conductance voltage- and Ca²⁺-activated K⁺ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

Abstract

Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

Central role of the BK channel in urinary bladder smooth muscle physiology and pathophysiology

“…In contrast, the latter depends mainly on Ca 2ϩ entry through L-type VDCCs and not Ca 2ϩ release via RyRs as pharmacological inhibition of L-type VDCCs substantially reduces the steady-state BK current (53). Thus, ryanodine has been largely used as a pharmacological tool to separate the TBKCs from the steady-state BK currents in UBSM cells (53,60,62,114,121,152).…”

“…Although BK channel regulation by PKC has been known in other cell types for quite some time (128,141), it was not until very recently when it was shown that PKC can also regulate BK channels in UBSM (62,64). Specifically, in guinea pig UBSM cells, PKC inhibits BK channel activity indirectly via a Ca 2ϩ -dependent mechanism involving the attenuation of the Ca 2ϩ release through RyRs while increasing the global intracellular Ca 2ϩ necessary to activate UBSM contraction (62). Upon pharmacological activation, PKC could phosphorylate proteins in the SR such as RyRs and/or PLB (Fig.…”

“…This leads to an inhibition of Ca 2ϩ sparks and the associated TBKCs. BK channel inhibition causes UBSM cell membrane potential depolarization, activation of L-type VDCCs, an increase in the global intracellular Ca 2ϩ concentration, and activation of UBSM contractility (62).…”

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

“…63 Indeed, a number of studies have revealed that PKC-dependent signaling pathways may play a significant role in the regulation of functions in UBSMCs. 1,37,64,65 An interaction of PKC with BK channels has been investigated at the molecular and functional level. 64,66 A plausible explanation for this interaction is that stimulation of M 3 Rs leads to diacylglycerol production, which activates PKC (Fig.…”

“…PKC may cause direct inhibition of the SR Ca 2C pumps and/or RyRs, thereby resulting in suppression of BK channels in UBSMCs. 64 However, the assumption of a direct PKC-BK channel interaction or an indirect mechanism involving the SR Ca 2C store needs to be tested directly. It has been shown that PKC can phosphorylate specific serine residues on the BK channel a and b subunits, and a failure of phosphorylation of either subunit leads to a loss of the channel function when the serine residues were substituted for alanine.…”