Pharmacological Opening of K V 7 Channels by the Novel Activator ML‐213: Role in Guinea Pig Urinary Bladder Smooth Muscle Function

It has been recently suggested that voltage‐gated K V 7 channels (K V 7.1‐K V 7.5) regulate urinary bladder smooth muscle (UBSM) function. Despite emerging developments, the physiological role of individual K V 7 channel subtypes remains less clear. Here, we utilized the novel compound N‐(2,4,6‐Trimethylphenyl)‐bicyclo[2.2.1]heptane‐2‐carboxamide (ML‐213), a potent activator of K V 7.2, K V 7.4, and K V 7.5 channels, to elucidate their physiological roles in guinea pig UBSM function. Using isometric UBSM tension recordings, Ca 2+ imaging, and amphotericin‐B perforated patch‐clamp electrophysiology, we elucidated the role of ML‐213‐sensitive K V 7 channels in regulating UBSM excitability and contractility. In functional studies of UBSM contractility, ML‐213 concentration‐dependently (100 nM‐30 μM) inhibited spontaneous phasic, pharmacologically‐induced, and nerve‐evoked contractions in UBSM isolated strips. In UBSM strips loaded with the ratiometric fluorescence probe fura 2, ML‐213 (10 μM) decreased the global intracellular Ca 2+ concentration and inhibited spontaneous Ca 2+ transients, which is consistent with the inhibitory effects on UBSM contractility. ML‐213‐induced attenuation of global Ca 2+ levels was abolished in the presence of the L‐type voltage‐gated Ca 2+ channel inhibitor nifedipine (1 μM) and the K V 7.1–K V 7.5 channel inhibitor XE991 (10 μM). These data suggests that ML‐213 decreases the global intracellular Ca 2+ concentration by inhibiting L‐type voltage‐gated Ca 2+ channels through an indirect mechanism downstream from K V 7 channel activation. In current‐clamp mode of the perforated patch‐clamp technique, ML‐213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in UBSM cells. ML‐213‐induced hyperpolarization of the UBSM cell membrane potential was reversible by washout of the compound. We next aimed to examine the effects of ML‐213 on voltage‐step depolarization‐induced whole cell K V 7 currents using the perforated patch‐clamp technique in voltage‐clamp mode. To isolate K V 7 currents, the extracellular bath solution contained the large conductance voltage‐ and Ca 2+ ‐activated K + channel inhibitor paxilline (1 μM) and gadolinium chloride (GdCl 3 , 100 μM), which blocks L‐type voltage‐gated Ca 2+ channels and non‐selective cation channels. Under these experimental conditions, ML‐213 (10 μM) enhanced whole cell K V 7 currents. These findings suggest that the modulation of K + transport through ML‐213‐sensitive K V 7 channels underlies ML‐213‐induced cell membrane hyperpolarization to decrease the global intracellular Ca 2+ concentration and UBSM contractility. These combined results, using the novel compound ML‐213, suggest that K V 7.2‐, K V 7.4‐, and K V 7.5‐containing channels are essential regulators of the excitability and contractility of UBSM by virtue of their control of the resting membrane potential. In addition, these studies provide a foundational basis for further studies investigating K V 7 channel functional roles in human UBSM excitability and contractility to confirm their potential as novel therapeutic targets for bladder dysfunction. Support or Funding Information Supported by NIH grant R01‐DK106964 to Georgi V. Petkov and F31‐DK104528 to Aaron Provence.