KV7 channel pharmacological activation by the novel activator ML213

Role for heteromeric KV7.4/KV7.5 Channels in Guinea pig detrusor smooth muscle function

Aaron Provence, Damiano Angoli, Georgi Petkov

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Voltage-gated KV7 channels (KV7.1 to KV7.5) 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 KV7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca21 imaging, amphotericin-B perforated patchclamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-Trimethylphenyl)- bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of KV7.2, KV7.4, and KV7.5 channels, to examine their physiologic roles in Guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 μM) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the KV7 channel inhibitor XE991 (10 μM). ML213 (0.1-30 μM) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 μM) decreased global intracellular Ca21 concentrations in Fura- 2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 μM) caused an increase in the amplitude of whole-cell KV7 currents. Further, in currentclamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 μM), consistent with ML213 activation of KV7 channel currents. Preapplication of XE991 (10 μM) not only depolarized the DSM cells, but also blocked ML213- induced hyperpolarization, confirming ML213 selectivity for KV7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric KV7.4/KV7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive KV7.4-And KV7.5-containing channels are essential regulators of DSM excitability and contractility.

Original languageEnglish (US)
Pages (from-to)131-144
Number of pages14
JournalJournal of Pharmacology and Experimental Therapeutics
Volume364
Issue number1
DOIs
StatePublished - Jan 1 2018

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Smooth Muscle
Guinea Pigs
Pharmacology
Smooth Muscle Myocytes
Electrophysiology
Membrane Potentials
Ligation
Cell Membrane
Muscle Tonus
Fura-2
Optical Imaging
Amphotericin B
Urinary Bladder

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

@article{4291014a53824aa491ba08ec267bfe9e,
title = "KV7 channel pharmacological activation by the novel activator ML213: Role for heteromeric KV7.4/KV7.5 Channels in Guinea pig detrusor smooth muscle function",
abstract = "Voltage-gated KV7 channels (KV7.1 to KV7.5) 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 KV7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca21 imaging, amphotericin-B perforated patchclamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-Trimethylphenyl)- bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of KV7.2, KV7.4, and KV7.5 channels, to examine their physiologic roles in Guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 μM) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the KV7 channel inhibitor XE991 (10 μM). ML213 (0.1-30 μM) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 μM) decreased global intracellular Ca21 concentrations in Fura- 2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 μM) caused an increase in the amplitude of whole-cell KV7 currents. Further, in currentclamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 μM), consistent with ML213 activation of KV7 channel currents. Preapplication of XE991 (10 μM) not only depolarized the DSM cells, but also blocked ML213- induced hyperpolarization, confirming ML213 selectivity for KV7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric KV7.4/KV7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive KV7.4-And KV7.5-containing channels are essential regulators of DSM excitability and contractility.",
author = "Aaron Provence and Damiano Angoli and Georgi Petkov",
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N2 - Voltage-gated KV7 channels (KV7.1 to KV7.5) 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 KV7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca21 imaging, amphotericin-B perforated patchclamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-Trimethylphenyl)- bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of KV7.2, KV7.4, and KV7.5 channels, to examine their physiologic roles in Guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 μM) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the KV7 channel inhibitor XE991 (10 μM). ML213 (0.1-30 μM) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 μM) decreased global intracellular Ca21 concentrations in Fura- 2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 μM) caused an increase in the amplitude of whole-cell KV7 currents. Further, in currentclamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 μM), consistent with ML213 activation of KV7 channel currents. Preapplication of XE991 (10 μM) not only depolarized the DSM cells, but also blocked ML213- induced hyperpolarization, confirming ML213 selectivity for KV7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric KV7.4/KV7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive KV7.4-And KV7.5-containing channels are essential regulators of DSM excitability and contractility.

AB - Voltage-gated KV7 channels (KV7.1 to KV7.5) 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 KV7 channel function at the molecular, cellular, and tissue levels in combination with pharmacological tools. We used isometric DSM tension recordings, ratiometric fluorescence Ca21 imaging, amphotericin-B perforated patchclamp electrophysiology, and in situ proximity ligation assay (PLA) in combination with the novel compound N-(2,4,6-Trimethylphenyl)- bicyclo[2.2.1]heptane-2-carboxamide (ML213), an activator of KV7.2, KV7.4, and KV7.5 channels, to examine their physiologic roles in Guinea pig DSM function. ML213 caused a concentration-dependent (0.1-30 μM) inhibition of spontaneous phasic contractions in DSM isolated strips; effects blocked by the KV7 channel inhibitor XE991 (10 μM). ML213 (0.1-30 μM) also reduced pharmacologically induced and nerve-evoked contractions in DSM strips. Consistently, ML213 (10 μM) decreased global intracellular Ca21 concentrations in Fura- 2-loaded DSM isolated strips. Perforated patch-clamp electrophysiology revealed that ML213 (10 μM) caused an increase in the amplitude of whole-cell KV7 currents. Further, in currentclamp mode of the perforated patch clamp, ML213 hyperpolarized DSM cell membrane potential in a manner reversible by washout or XE991 (10 μM), consistent with ML213 activation of KV7 channel currents. Preapplication of XE991 (10 μM) not only depolarized the DSM cells, but also blocked ML213- induced hyperpolarization, confirming ML213 selectivity for KV7 channel subtypes. In situ PLA revealed colocalization and expression of heteromeric KV7.4/KV7.5 channels in DSM isolated cells. These combined results suggest that ML213-sensitive KV7.4-And KV7.5-containing channels are essential regulators of DSM excitability and contractility.

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