K7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric K7.4/K7.5 Channels in Guinea Pig Detrusor Smooth Muscle Function.

Voltage-gated K7 channels (K7.1 to K7.5) are important regulators of the cell membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. This study sought to further the current knowledge of K7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound -(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K7.2, K7.4, and K7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 M) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K7 channel inhibitor XE991 (10 M). ML213 (0.1-30 M) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 M) decreased global intracellular Ca concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 M) caused an increase in the amplitude of whole-cell K7 currents. Further, in current-clamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 M), consistent with ML213 activation of K7 channel currents. Preapplication of XE991 (10 M) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K7.4/K7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K7.4- and K7.5-containing channels are essential regulators of DSM excitability and contractility.