Voltage-gated K(V)7 channels (K(V)7.1 to K(V)7.5) 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 K(V)7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca2+ imaging, amphotericin-B perforated patch-clamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-trime-thylphenyl)- bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of K(V)7.2, K(V)7.4, and K(V)7.5 channels, to examine their physiologic roles in guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 mu M) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the K(V)7 channel inhibitor XE991 (10 mu M). ML213 (0.1-30 mu M) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 mu M) decreased global intracellular Ca2+ concentrations in Fura-2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 mu M) caused an increase in the amplitude of whole-cell K(V)7 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 mu M), consistent with ML213 activation of K(V)7 channel currents. Preapplication of XE991 (10 mu M) not only depolarized the DSM cells, but also blocked ML213-induced hyperpolarization, confirming ML213 selectivity for K(V)7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric K(V)7.4/K(V)7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive K(V)7.4- and K(V)7.5-containing channels are essential regulators of DSM excitability and contractility.

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.

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.

We recently reported key physiologic roles for Ca2+-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca2+-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca2+, inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the sarcoplasmic reticulum represents a potential Ca2+ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs 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 IP3Rs and are activated by IP3R-mediated Ca2+ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca2+-mediated TRPM4-IP3R regulatory mechanism. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP3Rs with the selective IP3R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP3Rs have a key role in mediating the Ca2+-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 IP3Rs are spatially and functionally coupled in human DSM.

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology III1 Apr 2017PD70-08 KV7 CHANNEL PHARMACOLOGICAL MODULATION IN HUMAN DETRUSOR: A PROMISING TWO-WAY STREET FOR THE POTENTIAL TREATMENT OF OVERACTIVE AND UNDERACTIVE BLADDER Aaron Provence, Damiano Angoli, Eric Rovner, and Georgi Petkov Aaron ProvenceAaron Provence More articles by this author , Damiano AngoliDamiano Angoli More articles by this author , Eric RovnerEric Rovner More articles by this author , and Georgi PetkovGeorgi Petkov More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.3162AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent studies on rodents suggest that voltage-gated KV7 channels (KV7.1-KV7.5) are functionally expressed in detrusor smooth muscle (DSM). Here, we sought to validate the KV7 channels as potential novel targets in the pharmacological treatment of overactive bladder and/or underactive bladder by elucidating their functional role in human freshly-isolated DSM cells and tissues. METHODS Human DSM tissues were collected from patient-donors undergoing routine open bladder surgeries in accordance with IRB protocol Pro00045232. Combined methodology including isometric DSM tension recordings, ratiometric fluorescence Ca2+ imaging, and perforated patch-clamp electrophysiology, was applied to ascertain the role of the KV7 channel subtypes in human DSM function. RESULTS The KV7 channel activator, retigabine, decreased global Ca2+ concentrations in DSM isolated strips, while the KV7 channel inhibitor XE991 increased global DSM Ca2+ concentrations. Retigabine decreased spontaneous phasic and nerve-evoked contractions in DSM isolated strips. On the contrary, XE991 increased spontaneous phasic and nerve-evoked contractions in DSM isolated strips. Retigabine-induced DSM relaxation was attenuated in the presence of XE991. The KV7.2/KV7.3 channel activator ICA-069673 and KV7.1 activator L-364,373 also inhibited DSM spontaneous phasic contractions. In freshly-isolated DSM cells, retigabine hyperpolarized the DSM cell membrane potential, while XE991 induced membrane depolarization. Consistent with retigabine, the novel and selective KV7.4/KV7.5 channel activator ML213, also hyperpolarized the DSM cell membrane potential. CONCLUSIONS Collectively, these data provide promising evidence for the potential therapeutic utility of selective KV7 channel pharmacological modulators. DSM KV7 channels appear to be a universal therapeutic switch for bladder dysfunction treatment. Pharmacological opening of KV7 channels may be an effective novel approach to treat overactive bladder, whereas KV7 channel inhibitors can potentially be used as novel therapeutics for underactive bladder. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1353 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Aaron Provence More articles by this author Damiano Angoli More articles by this author Eric Rovner More articles by this author Georgi Petkov More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2016MP28-17 SPATIAL AND FUNCTIONAL INTERACTIONS OF THE TRANSIENT RECEPTOR POTENTIAL MELASTATIN-4 CHANNELS AND INOSITOL TRISPHOSPHATE RECEPTORS: NOVEL REGULATORY MECHANISM IN HUMAN DETRUSOR SMOOTH MUSCLE FUNCTION Aaron Provence, Kiril Hristov, Eric Rovner, and Georgi V. Petkov Aaron ProvenceAaron Provence More articles by this author , Kiril HristovKiril Hristov More articles by this author , Eric RovnerEric Rovner More articles by this author , and Georgi V. PetkovGeorgi V. Petkov More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1067AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Transient receptor potential melastatin-4 (TRPM4) channels are emerging as key regulators of human detrusor smooth muscle (DSM) function. However, the cellular mechanisms regulating TRPM4 channel activity in human DSM is virtually unexplored. As a potential cellular mechanism regulating TRPM4 channel activity in human DSM, we have directed our attention to the inositol trisphosphate (IP3) receptors (IP3Rs), which facilitate the release of Ca2+ from the sarcoplasmic reticulum (SR). Since the TRPM4 channels are activated by Ca2+, IP3R-mediated Ca2+ release of the SR represents a potential Ca2+ source for TRPM4 channel activation. Using clinically-characterized human tissues, we investigated the molecular and functional interactions of the TRPM4 channels and IP3Rs in human DSM cells. METHODS In accordance with the approved IRB Pro00045232 of the Medical University of South Carolina, we examined human DSM tissues from 6 donor-patients. Using in situ proximity ligation assay (PLA) and amphotericin-B perforated patch-clamp electrophysiology, we sought to test the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release of the SR in human DSM. RESULTS As determined by in situ PLA, we demonstrated close co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a TRPM4-IP3R regulatory mechanism in human DSM function. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, the selective IP3R inhibitor xestospongin C significantly decreased TICCs. These findings suggest that the SR IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. CONCLUSIONS In conclusion, this study provides novel insight into the cellular mechanisms regulating TRPM4 channel activity in human DSM function. The study reveals that the TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM, information that is critical for further evaluating the potential role of the TRPM4 channels as novel therapeutic targets for overactive bladder. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e378 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Aaron Provence More articles by this author Kiril Hristov More articles by this author Eric Rovner More articles by this author Georgi V. Petkov More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Overactive bladder (OAB) is a highly prevalent and debilitating condition that lacks an effective therapeutic treatment. The contraction and relaxation of urinary bladder smooth muscle (UBSM) facilitates the voiding and storage of urine, the two main functions of the urinary bladder. Some forms of OAB have been directly linked to UBSM dysfunction. Thus, efforts to uncover unknown regulatory mechanisms in UBSM function are urgently needed. Among the potential novel targets for OAB, emerging evidence points to key roles for transient receptor potential melastatin 4 (TRPM4) channels in UBSM excitability and contractility. However, the cellular mechanisms regulating TRPM4 channel activity in human UBSM has been virtually unexplored. As a potential cellular mechanism regulating TRPM4 channel activity in UBSM, we have directed our attention to the inositol trisphosphate (IP 3 ) receptors (IP 3 Rs), which facilitate the release of Ca 2+ from the sarcoplasmic reticulum (SR). Since the TRPM4 channels are activated by Ca 2+ , IP 3 R‐mediated Ca 2+ release of the SR represents a potential Ca 2+ source for TRPM4 channel activation. In accordance with the approved IRB Pro00045232 of the Medical University of South Carolina, we used clinically‐characterized human UBSM tissues from 6 donor patients to investigate the molecular and functional interactions of the TRPM4 channels and IP 3 Rs in human UBSM. Using in situ proximity ligation assay (PLA) and amphotericin‐B perforated patch‐clamp electrophysiology, we sought to test the hypothesis that TRPM4 channels are tightly associated with the IP 3 Rs and are activated by IP 3 R‐mediated Ca 2+ release of the SR in human UBSM. As determined by in situ PLA, we demonstrated colocalization of the TRPM4 channels and IP 3 Rs in human UBSM cells. As the TRPM4 channels and IP 3 Rs must be located within close apposition to functionally interact, these findings support the concept of a TRPM4‐IP 3 R regulatory mechanism in human UBSM function. To investigate IP 3 R regulation of TRPM4 channel activity, we sought to determine the consequences of IP 3 R pharmacological inhibition on TRPM4 channel‐mediated transient inward cation currents (TICCs). In freshly‐isolated human UBSM cells, the selective IP 3 R inhibitor xestospongin C significantly decreased TICCs. These findings suggest that the SR IP 3 Rs have a key role in mediating the Ca 2+ ‐dependent activation of TRPM4 channels in human UBSM. In conclusion, this study provides novel insight into the cellular mechanisms regulating TRPM4 channel activity in human UBSM function. Our study reveals that the TRPM4 channels and IP 3 Rs are spatially and functionally coupled in human UBSM, information that is critical for further evaluating the potential role of the TRPM4 channels as novel therapeutic targets for OAB. Support or Funding Information Supported by NIH grant R01‐DK106964 to Georgi V. Petkov and F31‐DK104528 to Aaron Provence.

Objective Our recent studies have demonstrated voltage-gated KCNQ channels (KCNQ1-KCNQ5) as key regulators of detrusor smooth muscle (DSM) function. Despite emerging developments, the physiological role of individual KCNQ channel subtypes remains less clear. Here, we utilized the novel compound ML-213, a potent activator of KCNQ2, KCNQ4, and KCNQ5 channels, to elucidate their physiological roles in guinea pig DSM function. Methods Using isometric DSM tension recordings, Ca2+ imaging, and amphotericin-B perforated patch-clamp electrophysiology, we elucidated the role of ML-213-sensitive KCNQ channels in regulating DSM excitability and contractility. Results ML-213 concentration-dependently (100 nM–30 µM) inhibited spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in DSM isolated strips. ML-213 (10 µM) decreased the global intracellular Ca2+ concentrations in DSM isolated strips, effects blocked by the L-type voltage-gated Ca2+ (CaV) channel inhibitor nifedipine (1 µM) and the KCNQ1-KCNQ5 channel inhibitor XE991 (10 µM). These data suggest that ML-213 decreases the global intracellular Ca2+ concentration by inhibiting L-type CaV channels through an indirect mechanism downstream from KCNQ channel activation. In addition, ML-213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in DSM cells, effects reversible by washout. We next aimed to examine the effects of ML-213 on whole cell KCNQ currents. To isolate KCNQ currents, the bath solution contained the large conductance voltage- and Ca2+-activated K+ channel inhibitor paxilline (1 µM) and gadolinium chloride (GdCl3, 50 µM), which blocks L-type CaV channels and non-selective cation channels. Under these experimental conditions, ML-213 (10 µM) enhanced whole cell KCNQ currents. These findings suggest that the modulation of K+ transport through ML-213-sensitive KCNQ channels underlies ML-213-induced cell membrane hyperpolarization to decrease the global intracellular Ca2+ concentration and DSM contractility. Conclusions These data using the novel compound ML-213, suggest that KCNQ2-, KCNQ4-, and KCNQ5-containing channels are essential regulators of the excitability, intracellular Ca2+ concentration, and contractility of DSM by virtue of their control of the membrane potential. Moreover, these new findings provide a foundational basis for future investigations on KCNQ channel functional roles in human DSM excitability and contractility to confirm their potential as novel therapeutic targets for overactive bladder. Source of Funding NIH grant R01-DK106964 to GV Petkov and F31-DK104528 to A Provence.

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2016MP28-18 17β-ESTRADIOL DIRECT ACTIVATION OF LARGE CONDUCTANCE VOLTAGE- AND CA2+-ACTIVATED K+ CHANNELS: NOVEL REGULATORY MECHANISM IN HUMAN DETRUSOR SMOOTH MUSCLE Kiril Hristov, Shankar Parajuli, Aaron Provence, Eric Rovner, and Georgi Petkov Kiril HristovKiril Hristov More articles by this author , Shankar ParajuliShankar Parajuli More articles by this author , Aaron ProvenceAaron Provence More articles by this author , Eric RovnerEric Rovner More articles by this author , and Georgi PetkovGeorgi Petkov More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1068AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Estrogens are key modulators of detrusor smooth muscle (DSM) function. However, the mechanism by which estrogens regulate DSM excitability and contractility in humans is not fully understood. Here, we examined the role of the large conductance voltage- and Ca2+-activated K+ (BK) channels in mediating the effects of estrogens in human DSM. METHODS We used freshly-isolated DSM cells and tissue strips from 20 patients without preoperative history of overactive bladder to examine the physiological role of 17β-estradiol in regulating DSM function. We employed amphotericin-B perforated patch-clamp, single BK channel recordings, and isometric DSM tension recordings in human DSM isolated strips. RESULTS In human DSM isolated strips, 17β-Estradiol (100 nM-1 μM) inhibited DSM spontaneous phasic contractions in a concentration-dependent manner (n=5). At 1 μM 17β-estradiol significantly inhibited contraction amplitude and muscle force integral by 53.3±8.9% and 44.35±13.2%, respectively (n=5). These 17β-estradiol inhibitory effects were blocked by 1 μM paxilline, a BK channel inhibitor (n=3). 17β-Estradiol (100 nM) also inhibited nerved-evoked contractions induced by electrical fiend stimulation (3-50 Hz). Patch-clamp experiments showed that 17β-estradiol (100 nM) increased depolarization-induced steady-state whole cell BK currents in human DSM cells. At the highest recording voltage of +80 mV, the whole cell BK currents were 33.9±6.5 and 44.5±7.8 pA/pF in the absence and presence of 100 nM 17β-estradiol, respectively (n=10). These 17β-estradiol stimulatory effects were blocked by 1 μM paxilline (n=4). At a holding potential of -20 mV human DSM cells exhibited transient BK currents (TBKCs). 17β-Estradiol increased TBKC frequency by 74.6±37.8% (n=8). Furthermore, 17β-estradiol (100 nM) hyperpolarized the DSM cell membrane potential by ≈ 2.5 mV (n=9). In inside-out excised patches, 17β-estradiol (100 nM) increased the single BK channel open probability by ≈ 60% (n=9), supporting the concept that 17β-estradiol activates BK channels directly rather than by an indirect genomic mechanism. CONCLUSIONS Our study reveals that at nanomolar physiological concentrations 17β-estradiol activates BK channels in human DSM via a direct non-genomic mechanism. 17β-Estradiol-induced BK channel activation leads to DSM cell membrane hyperpolarization and inhibition of the spontaneous phasic and nerve-evoked contractions in human DSM. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e378 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Kiril Hristov More articles by this author Shankar Parajuli More articles by this author Aaron Provence More articles by this author Eric Rovner More articles by this author Georgi Petkov More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2016MP30-05 SELECTIVE PHARMACOLOGICAL ACTIVATION OF INDIVIDUAL KCNQ CHANNEL SUBTYPES IN DETRUSOR SMOOTH MUSCLE: PROMISING NOVEL APPROACH FOR OVERACTIVE BLADDER TREATMENT Aaron Provence, Kiril Hristov, and Georgi V. Petkov Aaron ProvenceAaron Provence More articles by this author , Kiril HristovKiril Hristov More articles by this author , and Georgi V. PetkovGeorgi V. Petkov More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1237AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It has been recently suggested that voltage-gated KCNQ channels (KCNQ1-KCNQ5) regulate detrusor smooth muscle (DSM) function. Despite emerging developments, the physiological role of individual KCNQ channel subtypes remains less clear. Here, we utilized the novel compound ML-213, a potent activator of KCNQ2, KCNQ4, and KCNQ5 channels, to elucidate their physiological roles in DSM function. METHODS Using isometric DSM tension recordings, Ca2+ imaging, and amphotericin-B perforated patch-clamp electrophysiology, we elucidated the role of ML-213-sensitive KCNQ channels in regulating guinea pig DSM excitability and contractility. RESULTS ML-213 concentration-dependently (100 nM-30 µM) inhibited spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in DSM isolated strips. ML-213 (10 µM) decreased the global intracellular Ca2+ concentration and inhibited spontaneous Ca2+ transients, effects blocked by the L-type voltage-gated Ca2+ (CaV) channel inhibitor nifedipine (1 µM) and the KCNQ1- KCNQ5 channel inhibitor XE991 (10 µM). These data suggests that ML-213 decreases the global intracellular Ca2+ concentration by inhibiting L-type CaV channels through an indirect mechanism downstream from KCNQ channel activation. In addition, ML-213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in DSM cells, effects reversible by washout. We next aimed to examine the effects of ML-213 on whole cell KCNQ currents. To isolate KCNQ currents, the bath solution contained the large conductance voltage- and Ca2+-activated K+ channel inhibitor paxilline (1 µM) and gadolinium chloride (GdCl3, 100 µM), which blocks L-type CaV channels and non-selective cation channels. Under these experimental conditions, ML-213 (10 µM) enhanced whole cell KCNQ currents. These findings suggest that the modulation of K+ transport through ML-213-sensitive KCNQ channels underlies ML-213-induced cell membrane hyperpolarization to decrease the global intracellular Ca2+ concentration and DSM contractility. CONCLUSIONS These results, using the novel KCNQ channel opener ML-213, suggest that KCNQ2-, KCNQ4-, and KCNQ5-containing channels are essential regulators of the excitability and contractility of DSM, and therefore could represent novel molecular targets for pharmacological or genetic control of overactive bladder. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e412-e413 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Aaron Provence More articles by this author Kiril Hristov More articles by this author Georgi V. Petkov More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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.

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 Ca2+-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 Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ 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.

We examined the role of the large conductance voltage- and Ca2+-activated K+ (BK) channels as non-genomic targets for 17β-estradiol in guinea pig urinary bladder smooth muscle (UBSM). We performed single BK channel recordings on inside-out excised membrane patches and amphotericin-B perforated whole cell patch-clamp in combination with the BK channel inhibitor paxilline to determine the role of the BK channels as direct non-genomic targets for 17β-estradiol in UBSM cells. 17β-estradiol (100 nM) significantly increased the amplitude of depolarization-induced steady-state whole cell K+ currents and the frequency of transient BK currents in UBSM cells. The stimulatory effects on whole cell K+ currents by 17β-estradiol were blocked by the BK channel inhibitor paxilline (1 µM). 17β-estradiol (100 nM) significantly increased the single BK channel open probability, indicating direct activation of the BK channels. 17β-estradiol (100 nM) caused a significant hyperpolarization of the resting membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline (1 µM). 17β-estradiol (100 nM) had no inhibitory effects on L-type voltage-gated Ca2+ channels. These data support a critical role for the BK channels as direct non-genomic targets for 17β-estradiol in UBSM cells, thus regulating UBSM excitability and contractility. Supported by NIH R01-DK04284 to Georgi V. Petkov & NIH F31DK104528 to Aaron Provence.

We elucidated the physiological role of KV7.2/KV7.3 channels in guinea pig urinary bladder smooth muscle (UBSM) in vitro using the novel KV7.2/KV7.3 channel opener ICA-069673 and a multidisciplinary experimental approach. Western blot revealed protein expression for KV7.2 and KV7.3 channels in UBSM. In isolated UBSM cells, immunocytochemistry detected protein expression of KV7.2 and KV7.3 channels with localization at the cell membrane. ICA-069673 caused a concentration-dependent (100 nM-30 µM) inhibition of spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in UBSM isolated strips. In solution with elevated K+ concentration (60 mM), the inhibitory effects of ICA-069673 on UBSM contractility were attenuated. ICA-069673 decreased the global intracellular Ca2+ concentration in UBSM cells, an effect blocked by the L-type voltage-gated Ca2+ channel inhibitor nifedipine (1 µM). ICA-069673 significantly hyperpolarized the membrane potential and inhibited spontaneous action potentials in UBSM cells measured with perforated patch-clamp. XE991 (10 µM), a Kv7 channel inhibitor, abolished the effects of ICA069673 on USBM contractility and membrane potential. These results establish KV7.2/KV7.3 channels as critical regulators of UBSM excitability and contractility. Supported by NIH R01DK04284 to Georgi V. Petkov & NIH F31DK104528 to Aaron Provence.

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology I1 Apr 2015PD7-09 VOLTAGE-GATED KCNQ CHANNELS IN HUMAN DETRUSOR SMOOTH MUSCLE CONTRACTILITY: A NOVEL TARGET FOR THE PHARMACOLOGICAL TREATMENT OF OVERACTIVE BLADDER Aaron Provence, Kiril L. Hristov, Shankar P. Parajuli, Eric S. Rovner, and Georgi V. Petkov Aaron ProvenceAaron Provence More articles by this author , Kiril L. HristovKiril L. Hristov More articles by this author , Shankar P. ParajuliShankar P. Parajuli More articles by this author , Eric S. RovnerEric S. Rovner More articles by this author , and Georgi V. PetkovGeorgi V. Petkov More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.912AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent studies in rodents indicate critical roles for voltage-gated K+ (KCNQ) channels in detrusor smooth muscle (DSM) excitability and contractility. However, their role in human DSM function remains unknown. Here, we sought to investigate the KCNQ channel expression and function in human DSM and pharmacologically characterize the functional role of KCNQ channels in human DSM contractility in vitro. METHODS RT-PCR experiments were used to detect mRNA transcripts for KCNQ channel subtypes (KCNQ1-5). We further investigated KCNQ channel protein expression using Western blot analysis. Isometric DSM tension recordings were conducted to determine the functional role of the KCNQ channel subtypes in human DSM contractility. Human DSM tissues were collected from open bladder surgeries and represent patients without preoperative symptoms of overactive bladder (OAB). RESULTS In human DSM tissue, RT-PCR detected mRNA transcripts for all KCNQ channel subtypes except KCNQ2, however only KCNQ4 and KCNQ5 channel mRNA was detected in human DSM single cells (n=3). Western blot analysis detected protein expression for all KCNQ channel subtypes in human DSM tissue (n=3-4). In functional studies of human DSM contractility, the KCNQ channel inhibitor XE991 significantly increased DSM spontaneous phasic and nerve-evoked contractions (n=10-12). The KCNQ channel activators retigabine (KCNQ2-5 activator), ICA-069673 (KCNQ2-3 activator), and L-364,373 (KCNQ1 activator) decreased spontaneous phasic and nerve evoked contractions in human DSM isolated strips (n=4-12). The inhibitory effects of retigabine on DSM spontaneous phasic contractions were abolished when the KCNQ channels are inhibited with XE991 (n=5). CONCLUSIONS Our combined results suggest that pharmacological activation or blockade of KCNQ channels can affect human DSM contractility in vitro. These findings support further investigation of these channels and their role in normal and abnormal lower urinary tract function and their potential role as novel pharmacological targets for the treatment of OAB. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e188 Peer Review Report Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Aaron Provence More articles by this author Kiril L. Hristov More articles by this author Shankar P. Parajuli More articles by this author Eric S. Rovner More articles by this author Georgi V. Petkov More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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 novel K(V)7.2/K(V)7.3 channel activator ICA-069673 [N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide]. We employed a multilevel experimental approach using Western blot analysis, immunocytochemistry, isometric DSM tension recordings, fluorescence Ca2+ 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+ (60 mM), the effects of ICA-069673 on DSM tonic contractions were significantly attenuated. ICA-069673 decreased the global intracellular Ca2+ concentration in DSM cells, an effect blocked by the L-type Ca2+ 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.