K V 7 Channel Subtype Expression and Physiological Roles in Rat Urinary Bladder Smooth Muscle Excitability and Contractility

Abstract

Introduction Voltage-gated K+ (KV) channels, specifically type 7 (KV7), are emerging key regulators of urinary bladder smooth muscle (UBSM) excitability and contractility. The KV7 channel family consists of 5 pore-forming α-subunits (KV7.1–KV7.5) capable of assembling as homotetrameric (e.g. KV7.4) or heterotetrameric (e.g. KV7.4/KV7.5) channels; thus, further increasing species/tissue expression diversity and regulatory potential. However, much remains unknown about KV7 channel properties in UBSM and the translational value of the common experimental animal model - the rat. Hypothesis KV7 channels, especially homomeric or heteromeric KV7.4 and KV7.5 subtypes, regulate rat UBSM excitability and contractility. Methods UBSM tissue without mucosa was obtained from adult male Sprague Dawley rats. UBSM homogenates were used for Western blots to detect KV7 channel subtypes with or without surface biotinylation. UBSM isometric tension recordings were carried out in the presence of tetrodotoxin for spontaneous, 20 mM K+-induced and 1 µM carbachol-induced contractions or in its absence for the electrical field stimulated (EFS) contractions. For electrophysiological amphotericin-B perforated patch-clamp studies, single UBSM cells were prepared using enzymatic dissociation. UBSM cell membrane potential was recorded in current-clamp (I=0) mode at room temperature. The following KV7 channel modulators were tested: XE991 (KV7.1-KV7.5 subtype blocker), retigabine (KV7.2-KV7.5 activator), and ICA-069673 (selective activator for KV7.2/KV7.3, which at higher concentrations preferentially enhances KV7.4 and KV7.4/KV7.5 but not KV7.5 channels). Results Western blot experiments detected KV7.4 and KV7.5 channels in UBSM under both non- and surface-biotinylation assay conditions. The latter condition revealed that for both KV7.4 and KV7.5 subtypes surface localization predominated (>75 %). In isometric tension experiments, retigabine attenuated contractility in a concentration-dependent manner for all four contraction protocols (EC50 values: 2.6-58.7 µM for the amplitude and force, with complete inhibition of contractility at 100 µM, n=3-10). XE991 (10 µM) depolarized the membrane potential of isolated UBSM cells (Δ10.5±3.6 mV, n=5, p<0.05). In contrast, retigabine (10 µM, Δ-6.5±1.8 mV, n=4, p<0.04) and ICA-069673 (10 µM, Δ-11.5±2.4 mV, n=4, p<0.02), when applied separately, induced UBSM cell hyperpolarization. In the presence of XE991, ICA-069673 (10 µM) did not significantly change the membrane potential of isolated UBSM cells (Δ0.4±1.2 mV, n=4, p>0.05). Conclusions Our data show that both KV7.4 and KV7.5 channel subtypes are expressed in rat UBSM and display preferential cell membrane localization. Pharmacological modulation of KV7 channels can control UBSM cell excitability and tissue contractility. The hyperpolarizing effect of ICA-069673, which is blocked by XE991, revealed a critical role of KV7.4 homomeric and/or KV7.4/KV7.5 heteromeric channels in UBSM function.