The Novel and Selective KV7 Channel Activator, ML213, Reveals Key Physiological Roles for KV7.4 and KV7.5 Channel Subtypes in Guinea Pig Detrusor Smooth Muscle Function

Abstract

Voltage‐gated KV7 channels (KV7.1–KV7.5) are involved in establishing and maintaining the resting membrane potential in detrusor smooth muscle (DSM) of the urinary bladder. Here, we utilized isometric DSM tension recordings, ratiometric fluorescence Ca2+ imaging, and perforated patch‐clamp electrophysiology in combination with the novel compound N‐(2,4,6‐Trimethylphenyl)‐bicyclo[2.2.1]heptane‐2‐carboxamide (ML213), a potent activator of KV7.4 and KV7.5 channels, to dissect the functional roles of KV7.4 and KV7.5‐containing channels in guinea pig DSM excitability and contractility. ML213 concentration‐dependently (0.1–30 μM) inhibited spontaneous phasic contractions in DSM isolated strips, effects blocked by the KV7 channel inhibitor XE991 (10 μM). ML213 (0.1–30 μM) also inhibited DSM 20 mM KCl‐induced DSM phasic contractions. ML213 (10 μM) decreased the 60 mM KCl‐induced tonic contractions by 21.2% of the control, however, subsequent application of the L‐type Ca2+ channel inhibitor nifedipine (10 μM) caused a significantly higher inhibitory effect, decreasing DSM tone to 58.2% of the control. ML213 (0.1–30 μM)‐induced relaxation of DSM isolated strips was attenuated in a concentration‐dependent manner by the muscarinic receptor agonist carbachol (0.1–1 μM). ML213 concentration‐dependently (0.1–30 μM) inhibited nerve‐evoked contractions induced by electrical field stimulation (EFS) at continuous 10 Hz and 20 Hz as well as extended 0.5–50 Hz frequencies. ML213 (0.1–30 μM) had reduced inhibitory effects on 20 Hz EFS‐induced DSM contractions compared to 10 Hz EFS. Consistently, ML213 (10 μM) decreased global Ca2+ concentrations in fura‐2 loaded DSM isolated strips, effects blocked by carbachol (1 μM). Next, we examined ML213‐induced effects on the activity of KV7.4‐ and KV7.5‐containing channels using the perforated patch‐clamp technique. To effectively isolate KV7 currents, voltage‐clamp recordings were made in the presence of the selective BK channel inhibitor paxilline (1 μM) and GdCl3 (50 μM), an inhibitor of non‐selective cation currents. ML213 (10 μM) caused a significant increase in the amplitude of the KV7 currents, effects reversible by washout of ML213. In current‐clamp mode, we found that ML213 hyperpolarized the cell membrane potential, consistent with the potentiation of KV7 channel currents. ML213‐induced hyperpolarization of the DSM cell membrane potential was reversible by washout. These results suggest ML213‐sensitive KV7 channels serve an inhibitory functional role in DSM excitability and contractility. Further, ML213‐sensitive KV7 channels appear to function in opposition to cholinergic signaling pathways through a mechanism involving indirect modulation of L‐type Ca2+ channel activity downstream from pharmacologically activated KV7 channels. The combined results, using the novel compound ML213, suggest that ML213‐sensitive KV7.4‐ and KV7.5‐containing channels are essential regulators of DSM function.Support or Funding InformationSupported by NIH R01‐DK106964 to G. V. Petkov and F31‐DK104528 to A. Provence.