Voltage-gated K7 channels (K7.1 to K7.5) 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 K7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound -(2,4,6-trimethylphenyl)-bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K7.2, K7.4, and K7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 M) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K7 channel inhibitor XE991 (10 M). ML213 (0.1-30M) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 M) decreased global intracellular Ca concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 M) caused an increase in the amplitude of whole-cell K7 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 M), consistent with ML213 activation of K7 channel currents. Preapplication of XE991 (10 M) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K7.4/K7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K7.4- and K7.5-containing channels are essential regulators of DSM excitability and contractility.

The physiologic roles of voltage-gated K V 7 channel subtypes (K V 7.1-K V 7.5) in detrusor smooth muscle (DSM) are poorly understood. Here, we sought to elucidate the functional roles of K V 7.2/K V 7.3 channels in guinea pig DSM excitability and contractility using the novelWe employed a multilevel experimental approach using Western blot analysis, immunocytochemistry, isometric DSM tension recordings, fluorescence Ca 21 imaging, and perforated whole-cell patch-clamp electrophysiology. Western blot experiments revealed the protein expression of K V 7.2 and K V 7.3 channel subunits in DSM tissue. In isolated DSM cells, immunocytochemistry with confocal microscopy further confirmed protein expression for K V 7.2 and K V 7.3 channel subunits, where they localize within the vicinity of the cell membrane. ICA-069673 inhibited spontaneous phasic, pharmacologically induced, and nerve-evoked contractions in DSM isolated strips in a concentration-dependent manner. The inhibitory effects of ICA-069673 on DSM spontaneous phasic and tonic contractions were abolished in the presence of the K V 7 channel inhibitor XE991 [10,10-bis(4-pyridinylmethyl)-9 (10H)-anthracenone dihydrochloride]. Under conditions of elevated extracellular K 1 (60 mM), the effects of ICA-069673 on DSM tonic contractions were significantly attenuated. ICA-069673 decreased the global intracellular Ca 21 concentration in DSM cells, an effect blocked by the L-type Ca 21 channel inhibitor nifedipine. ICA-069673 hyperpolarized the membrane potential and inhibited spontaneous action potentials of isolated DSM cells, effects that were blocked in the presence of XE991. In conclusion, using the novel K V 7.2/ K V 7.3 channel activator ICA-069673, this study provides strong evidence for a critical role for the K V 7.2-and K V 7.3-containing channels in DSM function at both cellular and tissue levels.

Malysz J, Afeli SA, Provence A, Petkov GV. Ethanol-mediated relaxation of guinea pig urinary bladder smooth muscle: involvement of BK and L-type Ca 2ϩ channels. Am J Physiol Cell Physiol 306: C45-C58, 2014. First published October 23, 2013 doi:10.1152/ajpcell.00047.2013.-Mechanisms underlying ethanol (EtOH)-induced detrusor smooth muscle (DSM) relaxation and increased urinary bladder capacity remain unknown. We investigated whether the large conductance Ca 2ϩ -activated K ϩ (BK) channels or L-type voltage-dependent Ca 2ϩ channels (VDCCs), major regulators of DSM excitability and contractility, are targets for EtOH by patch-clamp electrophysiology (conventional and perforated whole cell and excised patch single channel) and isometric tension recordings using guinea pig DSM cells and isolated tissue strips, respectively. EtOH at 0.3% vol/vol (ϳ50 mM) enhanced whole cell BK currents at ϩ30 mV and above, determined by the selective BK channel blocker paxilline. In excised patches recorded at ϩ40 mV and ϳ300 nM intracellular Ca 2ϩ concentration ([Ca 2ϩ ]), EtOH (0.1-0.3%) affected single BK channels (mean conductance ϳ210 pS and blocked by paxilline) by increasing the open channel probability, number of open channel events, and open dwell-time constants. The amplitude of single BK channel currents and unitary conductance were not altered by EtOH. Conversely, at ϳ10 M but not ϳ2 M intracellular [Ca 2ϩ ], EtOH (0.3%) decreased the single BK channel activity. EtOH (0.3%) affected transient BK currents (TBKCs) by either increasing frequency or decreasing amplitude, depending on the basal level of TBKC frequency. In isolated DSM strips, EtOH (0.1-1%) reduced the amplitude and muscle force of spontaneous phasic contractions. The EtOH-induced DSM relaxation, except at 1%, was attenuated by paxilline. EtOH (1%) inhibited L-type VDCC currents in DSM cells. In summary, we reveal the involvement of BK channels and L-type VDCCs in mediating EtOHinduced urinary bladder relaxation accommodating alcohol-induced diuresis. smooth muscle; patch-clamp; contractility; detrusor; alcohol URINARY BLADDER FUNCTION DEPENDS on the contractile status of detrusor smooth muscle (DSM) cells. DSM relaxation allows for bladder expansion during urine storage, whereas its contraction, coupled with the opening of the bladder sphincter, permits urine voiding (3). Multiple ion channels and receptors are expressed in DSM cells regulating bladder function. Among them, the large-conductance voltage-and Ca 2ϩ -activated K ϩ channels (also known as BK, Slo, or K Ca 1.1 channels) and L-type voltage-dependent Ca 2ϩ channels (VDCCs) are recognized as key regulators of excitability and contractility of DSM (44). Supporting data, provided for DSM studies using rat, guinea pig, mouse, and importantly human tissues and cells (5,17,18,22,24,25,46), demonstrate the role of BK channels in the regulation of the resting membrane potential, modulation of the repolarization phase of DSM action potentials, and generation of spontaneous transient BK currents (TBKCs)...

Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca2+-activated K+ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca2+ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability.

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Estrogens have an important role in regulating detrusor smooth muscle (DSM) function. However, the underlying molecular and cellular mechanisms by which estrogens control human DSM excitability and contractility are not well known. Here, we used human DSM specimens from open bladder surgeries on 27 patients to elucidate the mechanism by which 17β‐estradiol regulates large conductance voltage‐ and Ca2+‐activated K+ (BK) channels, the most prominent K+ channels in human DSM. We employed single BK channel recordings on inside‐out excised membrane patches, perforated whole‐cell patch‐clamp on freshly isolated DSM cells, and isometric tension recordings on DSM‐isolated strips to investigate the mechanism by which 17β‐estradiol activates BK channels. 17β‐Estradiol (100 nmol/L) rapidly increased depolarization‐induced whole‐cell K+ currents in DSM cells. The 17β‐estradiol stimulatory effects on whole‐cell BK currents were completely abolished by the selective BK channel inhibitor paxilline (1 μmol/L), clearly indicating that 17β‐estradiol specifically activates BK channels. 17β‐Estradiol also increased the frequency of ryanodine receptor‐mediated transient BK currents. Single BK channel recordings showed that 17β‐estradiol (100 nmol/L) significantly increased the BK channel open probability of inside‐out excised membrane patches, revealing that 17β‐estradiol activates BK channels directly. 17β‐Estradiol reduced spontaneous phasic contractions of human DSM‐isolated strips in a concentration‐dependent manner (100 nmol/L‐1 μmol/L), and this effect was blocked by paxilline (1 μmol/L). 17β‐Estradiol (100 nmol/L) also reduced nerve‐evoked contractions of human DSM‐isolated strips. Collectively, our results reveal that 17β‐estradiol plays a critical role in regulating human DSM function through a direct nongenomic activation of BK channels.

We recently reported key physiologic roles for Ca-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca, inositol 1,4,5-trisphosphate receptor (IPR)-mediated Ca release from the sarcoplasmic reticulum represents a potential Ca source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IPRs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IPRs and are activated by IPR-mediated Ca release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IPRs in human DSM cells. As the TRPM4 channels and IPRs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca-mediated TRPM4-IPR regulatory mechanism. To investigate IPR regulation of TRPM4 channel activity, we sought to determine the consequences of IPR pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IPRs with the selective IPR inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IPRs have a key role in mediating the Ca-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IPRs are spatially and functionally coupled in human DSM.

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca-activated K (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of -20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability.