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K V 7 Channel Subtype Expression and Physiological Roles in Rat Urinary Bladder Smooth Muscle Excitability and Contractility
Author(s) -
Maxwell Sarah,
Malysz John,
Leo M. Dennis,
Xin Wenkuan,
Park Frank,
Petkov Georgi
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.04481
Subject(s) - contractility , channel blocker , medicine , membrane potential , electrophysiology , microbiology and biotechnology , endocrinology , biology , patch clamp , chemistry , biophysics , calcium
Voltage‐gated K + (K V ) channels, specifically type 7 (K V 7), are emerging key regulators of urinary bladder smooth muscle (UBSM) excitability and contractility. The K V 7 channel family consists of 5 pore‐forming α‐subunits (K V 7.1–K V 7.5) capable of assembling as homotetrameric (e.g. K V 7.4) or heterotetrameric (e.g. K V 7.4/K V 7.5) channels; thus, further increasing species/tissue expression diversity and regulatory potential. However, much remains unknown about K V 7 channel properties in UBSM and the translational value of the common experimental animal model ‐ the rat. Hypothesis K V 7 channels, especially homomeric or heteromeric K V 7.4 and K V 7.5 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 K V 7 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 K V 7 channel modulators were tested: XE991 (K V 7.1‐K V 7.5 subtype blocker), retigabine (K V 7.2‐K V 7.5 activator), and ICA‐069673 (selective activator for K V 7.2/K V 7.3, which at higher concentrations preferentially enhances K V 7.4 and K V 7.4/K V 7.5 but not K V 7.5 channels). Results Western blot experiments detected K V 7.4 and K V 7.5 channels in UBSM under both non‐ and surface‐biotinylation assay conditions. The latter condition revealed that for both K V 7.4 and K V 7.5 subtypes surface localization predominated ( > 75 %). In isometric tension experiments, retigabine attenuated contractility in a concentration‐dependent manner for all four contraction protocols (EC 50 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 K V 7.4 and K V 7.5 channel subtypes are expressed in rat UBSM and display preferential cell membrane localization. Pharmacological modulation of K V 7 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 K V 7.4 homomeric and/or K V 7.4/K V 7.5 heteromeric channels in UBSM function.