K_V7 Channel Pharmacological Activation by the Novel Activator ML213: Role for Heteromeric K_V7.4/K_V7.5 Channels in...

Voltage-gated K_V7 channels (K_V7.1 to K_V7.5) 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 K_V7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca²⁺ imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K_V7.2, K_V7.4, and K_V7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 µM) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K_V7 channel inhibitor XE991 (10 µM). ML213 (0.1-30 µM) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 µM) decreased global intracellular Ca²⁺ concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 µM) caused an increase in the amplitude of whole-cell K_V7 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 µM), consistent with ML213 activation of K_V7 channel currents. Preapplication of XE991 (10 µM) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K_V7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K_V7.4/K_V7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K_V7.4- and K_V7.5-containing channels are essential regulators of DSM excitability and contractility.