Search

Background and Purpose: T16A(inh) -A01 is a recently identified inhibitor of the calcium-activated chloride channel TMEM16A. The aim of this study was to test the efficacy of T16A(inh) -A01 for inhibition of calcium-activated chloride channels in vascular smooth muscle and consequent effects on vascular tone.Experimental Approach: Single channel and whole cell patch clamp was performed on single smooth muscle cells from rabbit pulmonary artery and mouse thoracic aorta. Isometric tension studies were performed on mouse thoracic aorta and mesenteric artery as well as human abdominal visceral adipose artery.Key Results: In rabbit pulmonary artery myocytes T16A(inh) -A01 (1-30 μM) inhibited single calcium (Ca(2+) )-activated chloride (Cl(-) ) channels and whole cell currents activated by 500 nM free Ca(2+) . Similar effects were observed for single Ca(2+) -activated Cl(-) channels in mouse thoracic aorta, and in both cell types, channel activity was abolished by two antisera raised against TMEM16A but not by a bestrophin antibody. The TMEM16A potentiator, F(act) (10 μM), increased single channel and whole cell Ca(2+) -activated Cl(-) currents in rabbit pulmonary arteries. In isometric tension studies, T16A(inh) -A01 relaxed mouse thoracic aorta pre-contracted with methoxamine with an IC(50) of 1.6 μM and suppressed the methoxamine concentration-effect curve. T16A(inh) -A01 did not affect the maximal contraction produced by 60 mM KCl and the relaxant effect of 10 μM T16A(inh) -A01 was not altered by incubation of mouse thoracic aorta in a cocktail of potassium (K(+) ) channel blockers. T16A(inh) -A01 (10 μM) also relaxed human visceral adipose arteries by 88 ± 3%.Conclusions and Implications: T16A(inh) -A01 blocks calcium-activated chloride channels in vascular smooth muscle cells and relaxes murine and human blood vessels. [ABSTRACT FROM AUTHOR]

Background and Purpose: The aim of this study was to characterize the functional impact of KCNQ1-encoded voltage-dependent potassium channels (K(v)7.1) in the vasculature.Experimental Approach: Mesenteric arteries, intrapulmonary arteries and thoracic aortae were isolated from adult rats. K(v)7.1 channel expression was established by fluorescence immunocytochemistry. Wire myography determined functionality of these channels in response to selective blockers and activators. Xenopus oocytes expressing K(v)7.1 channels were used to assess the effectiveness of selective K(v)7.1 channel blockers.Key Results: K(v)7.1 channels were identified in arterial myocytes by immunocytochemistry. K(v)7.1 blockers HMR1556, L-768,673 (10 µM) and JNJ39490282 (JNJ282; 1 µM) had no contractile effects in arteries, whereas the pan-K(v)7 channel blocker linopirdine (10 µM) evoked robust contractions. Application of two compounds purported to activate K(v)7.1 channels, L-364 373 (R-L3) and mefenamic acid, relaxed mesenteric arteries preconstricted by methoxamine. These responses were reversed by HMR1556 or L-768,673 but not JNJ282. Similar effects were observed in the thoracic aorta and intrapulmonary arteries.Conclusions and Implications: In contrast to previous assumptions, K(v)7.1 channels expressed in arterial myocytes are functional ion channels. Although these channels do not appear to contribute to resting vascular tone, K(v)7.1 activators were effective vasorelaxants. [ABSTRACT FROM AUTHOR]

KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to β-adrenoceptor-mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to β-adrenoceptor-mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired β-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension. [ABSTRACT FROM AUTHOR]

Background and Purpose: This study investigated the functional and electrophysiological effects of the Kv7 channel activator, retigabine, on murine portal vein smooth muscle.Experimental Approach: KCNQ gene expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical experiments. Whole cell voltage clamp and current clamp were performed on isolated myocytes from murine portal vein. Isometric tension recordings were performed on whole portal veins. K+ currents generated by KCNQ4 and KCNQ5 expression were recorded by two-electrode voltage clamp in Xenopus oocytes.Key Results: KCNQ1, 4 and 5 were expressed in mRNA derived from murine portal vein, either as whole tissue or isolated myocytes. Kv7.1 and Kv7.4 proteins were identified in the cell membranes of myocytes by immunocytochemistry. Retigabine (2-20 microM) suppressed spontaneous contractions in whole portal veins, hyperpolarized the membrane potential and augmented potassium currents at -20 mV. At more depolarized potentials, retigabine and flupirtine, decreased potassium currents. Both effects of retigabine were prevented by prior application of the K(v)7 blocker XE991 (10 muM). Recombinant KCNQ 4 or 5 channels were only activated by retigabine or flupirtine.Conclusions and Implications: The Kv7 channel activators retigabine and flupirtine have bimodal effects on vascular potassium currents, which are not seen with recombinant KCNQ channels. These results provide support for KCNQ4- or KCNQ5-encoded channels having an important functional impact in the vasculature. [ABSTRACT FROM AUTHOR]

Aims: Sodium/glucose transporter 2 (SGLT2 or SLC5A2) inhibitors lower blood glucose and are also approved treatments for heart failure independent of raised glucose. Various studies have showed that SGLT2 inhibitors relax arteries, but the underlying mechanisms are poorly understood and responses variable across arterial beds. We speculated that SGLT2 inhibitor-mediated arterial relaxation is dependent upon calcitonin gene-related peptide (CGRP) released from sensory nerves independent of glucose transport., Methods and Results: The functional effects of SGLT1 and 2 inhibitors (mizagliflozin, dapagliflozin, and empagliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted resistance arteries (mesenteric and cardiac septal arteries) as well as main renal conduit arteries from male Wistar rats using wire myography. SGLT2, CGRP, TRPV1, and NHE1 expression was determined by western blot and immunohistochemistry. Kv7.4/5/KCNE4 and TRPV1 currents were measured in the presence and absence of dapagliflozin and empagliflozin. All SGLT inhibitors (1-100 µM) and cariporide (30 µM) relaxed mesenteric arteries but had negligible effect on renal or septal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that were absent in renal and septal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin relaxed mesenteric arteries more effectively than renal or septal arteries. In mesenteric arteries, relaxations to dapagliflozin, empagliflozin, and cariporide were attenuated by the CGRP receptor antagonist BIBN-4096, depletion of sensory nerves with capsaicin, and blockade of TRPV1 or Kv7 channels. Neither dapagliflozin nor empagliflozin activated heterologously expressed TRPV1 channels or Kv7 channels directly. Sensory nerves also expressed NHE1 but not SGLT2 and cariporide pre-application as well as knockdown of NHE1 by translation stop morpholinos prevented the relaxant response to SGLT2 inhibitors., Conclusion: SGLT2 inhibitors relax mesenteric arteries by promoting the release of CGRP from sensory nerves in a NHE1-dependent manner., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Stimulation of the calcium-sensing receptor (CaSR) regulates vascular contractility, but cellular mechanisms involved remain unclear. This study investigated the role of perivascular sensory nerves in CaSR-induced relaxations of male rat mesenteric arteries. In fluorescence studies, colocalisation between synaptophysin, a synaptic vesicle marker, and the CaSR was present in the adventitial layer of arterial segments. Using wire myography, increasing external Ca 2+ concentration ([Ca 2+ ] o ) from 1 to 10 mM induced vasorelaxations, previously shown to involve the CaSR, which were inhibited by pretreatment with capsaicin. [Ca 2+ ] o -induced vasorelaxations were partially reduced by the calcitonin gene-related peptide (CGRP) receptor blockers, CGRP 8-37 and BIBN 4096, and the neurokinin 1 (NK1) receptor blocker L733,060. The inhibitory effect of CGRP 8-37 required a functional endothelium whereas the inhibitory action of L733,060 did not. Complete inhibition of [Ca 2+ ] o -induced vasorelaxations occurred when CGRP 8-37 and L733,060 were applied together. [Ca 2+ ] o -induced vasorelaxations in the presence of capsaicin were abolished by the ATP-dependent K + channel (K ATP ) blocker PNU 37883, but unaffected by the endothelium nitric oxide synthase (eNOS) inhibitor L-NAME. We suggest that the CaSR on perivascular sensory nerves mediate relaxations in rat mesenteric arteries via endothelium-dependent and -independent mechanisms involving CGRP and NK1 receptor-activated NO production and K ATP channels, respectively., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The KCNQ family is comprised of five genes and the expression products form voltage-gated potassium channels (Kv7.1-7.5) that have a major impact upon cellular physiology in many cell types. Each functional Kv7 channel forms as a tetramer that often associates with proteins encoded by the KCNE gene family (KCNE1-5) and is critically reliant upon binding of phosphatidylinositol bisphosphate (PIP 2 ) and calmodulin. Other modulators like A-kinase anchoring proteins, ubiquitin ligases and Ca-calmodulin kinase II alter Kv7 channel function and trafficking in an isoform specific manner. It has now been identified that for Kv7.4, G protein βγ subunits (Gβγ) can be added to the list of key regulators and is paramount for channel activity. This article provides an overview of this nascent field of research, highlighting themes and directions for future study., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Stott and Greenwood.)

Stimulation of the calcium-sensing receptor (CaSR) induces both vasoconstrictions and vasorelaxations but underlying cellular processes remain unclear. This study investigates expression and effect of stimulating the CaSR by increasing external Ca 2+ concentration ([Ca 2+ ] o ) on contractility of rat mesenteric arteries. Immunofluorescence studies showed expression of the CaSR in perivascular nerves, vascular smooth muscle cells (VSMCs), and vascular endothelium cells. Using wire myography, increasing [Ca 2+ ] o from 1 to 10 mM induced vasorelaxations which were inhibited by the calcilytic Calhex-231 and partially dependent on a functional endothelium. [Ca 2+ ] o -induced vasorelaxations were reduced by endothelial NO synthase (eNOS, L-NAME) and large conductance Ca 2+ -activated K + channels (BK Ca , iberiotoxin), with their inhibitory action requiring a functional endothelium. [Ca 2+ ] o -induced vasorelaxations were also markedly inhibited by an ATP-dependent K + channel (K ATP ) blocker (PNU37883), which did not require a functional endothelium to produce its inhibitory action. Inhibitor studies also suggested contributory roles for inward rectifying K + channels (K ir ), Kv7 channels, and small conductance Ca 2+ -activated K + channels (SK Ca ) on [Ca 2+ ] o -induced vasorelaxations. These findings indicate that stimulation of the CaSR mediates vasorelaxations involving multiple pathways, including an endothelium-dependent pathway involving NO production and activation of BK Ca channels and an endothelium-independent pathway involving stimulation of K ATP channels., (© 2024 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Sex hormones and the reproductive cycle (estrus in rodents and menstrual in humans) have a known impact on arterial function. In spite of this, sex hormones and the estrus/menstrual cycle are often neglected experimental factors in vascular basic preclinical scientific research. Recent research by our own laboratory indicates that cyclical changes in serum concentrations of sex -hormones across the rat estrus cycle, primary estradiol, have significant consequences for the subcellular trafficking and function of K V . Vascular potassium channels, including K V , are essential components of vascular reactivity. Our study represents a small part of a growing field of literature aimed at determining the role of sex hormones in regulating arterial ion channel function. This review covers key findings describing the current understanding of sex hormone regulation of vascular potassium channels, with a focus on K V channels. Further, we highlight areas of research where the estrus cycle should be considered in future studies to determine the consequences of physiological oscillations in concentrations of sex hormones on vascular potassium channel function.

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to Gq-protein receptor signaling. Such agonists stimulate oscillating calcium waves, the frequency of which drives the strength of contraction. These Ca2+ events are modulated by a variety of ion channels including voltage-gated calcium channels (CaV1.2), the Tmem16a or Anoctamin-1 (ANO1)-encoded calcium-activated chloride (CaCC) channel, and Ca2+ release from the sarcoplasmic reticulum through inositol-trisphosphate receptors (IP3R). Although these calcium events have been characterized, it is unclear how these calcium oscillations underly a sustained contraction in these muscle cells. We used smooth muscle-specific ablation of ANO1 and pharmacological tools to establish the role of ANO1, CaV1.2, and IP3R in the contractile and intracellular Ca2+ signaling properties of mouse PA smooth muscle expressing the Ca2+ biosensor GCaMP3 or GCaMP6. Pharmacological block or genetic ablation of ANO1 or inhibition of CaV1.2 or IP3R, or Ca2+ store depletion equally inhibited 5-HT-induced tone and intracellular Ca2+ waves. Coimmunoprecipitation experiments showed that an anti-ANO1 antibody was able to pull down both CaV1.2 and IP3R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both CaV1.2 and IP3R at or near the plasma membrane of PASMC from wild-type mice. We conclude that the stable 5-HT-induced PA contraction results from the integration of stochastic and localized Ca2+ events supported by a microenvironment comprising ANO1, CaV1.2, and IP3R. In this model, ANO1 and CaV1.2 would indirectly support cyclical Ca2+ release events from IP3R and propagation of intracellular Ca2+ waves., (© 2023 Akin et al.)

In addition, to their established role in cardiac myocytes and neurons, ion channels encoded by ether-a-go-go-related genes (ERG1-3 or kcnh2,3 and 6) (kcnh2) are functionally relevant in phasic smooth muscle. The aim of the study was to determine the expression and functional impact of ERG expression products in rat urinary bladder smooth muscle using quantitative polymerase chain reaction, immunocytochemistry, whole-cell patch-clamp and isometric tension recording. kcnh2 was expressed in rat bladder, whereas kcnh6 and kcnh3 expression were negligible. Immunofluorescence for the kcnh2 expression product Kv11.1 was detected in the membrane of isolated smooth muscle cells. Potassium currents with voltage-dependent characteristics consistent with Kv11.1 channels and sensitive to the specific blocker E4031 (1 μM) were recorded from isolated detrusor smooth muscles. Disabling Kv11.1 activity with specific blockers (E4031 and dofetilide, 0.2-20 μM) augmented spontaneous contractions to a greater extent than BK Ca channel blockers, enhanced carbachol-driven activity, increased nerve stimulation-mediated contractions, and impaired β-adrenoceptor-mediated inhibitory responses. These data establish for the first time that Kv11.1 channels are key determinants of contractility in rat detrusor smooth muscle., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Background and Purpose: Kcnq-encoded K V 7 channels (termed K V 7.1-5) regulate vascular smooth muscle cell (VSMC) contractility at rest and as targets of receptor-mediated responses. However, the current data are mostly derived from males. Considering the known effects of sex, the oestrous cycle and sex hormones on vascular reactivity, here we have characterised the molecular and functional properties of K V 7 channels from renal and mesenteric arteries from female Wistar rats separated into di-oestrus and met-oestrus (F-D/M) and pro-oestrus and oestrus (F-P/E)., Experimental Approach: RT-qPCR, immunocytochemistry, proximity ligation assay and wire myography were performed in renal and mesenteric arteries. Circulating sex hormone concentrations were determined by liquid chromatography-tandem mass spectrometry. Whole-cell electrophysiology was undertaken on cells expressing K V 7.4 channels in association with G-protein-coupled oestrogen receptor 1 (GPER1)., Key Results: The K V 7.2-5 activators S-1 and ML213 and the pan-K V 7 inhibitor linopirdine were more effective in arteries from F-D/M compared with F-P/E animals. In VSMCs isolated from F-P/E rats, exploratory evidence indicates reduced membrane abundance of K V 7.4 but not K V 7.1, K V 7.5 and Kcne4 when compared with cells from F-D/M. Plasma oestradiol was higher in F-P/E compared with F-D/M, and progesterone showed the converse pattern. Oestradiol/GPER1 agonist G-1 diminished K V 7.4 encoded currents and ML213 relaxations and reduced the membrane abundance of K V 7.4 and interaction between K V 7.4 and heat shock protein 90 (HSP90), in arteries from F-D/M but not F-P/E., Conclusions and Implications: GPER1 signalling decreased K V 7.4 membrane abundance in conjunction with diminished interaction with HSP90, giving rise to a 'pro-contractile state'., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background and Purpose: Prostacyclin mimetics express potent vasoactive effects via prostanoid receptors that are not unequivocally defined, as to date no study has considered sex as a factor. The aim of this study was to determine the contribution of IP and EP 3 prostanoid receptors to prostacyclin mimetic iloprost-mediated responses, whether K V 7.1-5 channels represent downstream targets of selective prostacyclin-IP-receptor agonist MRE-269 and the impact of the oestrus cycle on vascular reactivity., Experimental Approach: Within second-order mesenteric arteries from male and female Wistar rats, we determined (1) relative mRNA transcripts for EP 1-4 (Ptger 1-4 ), IP (Ptgi) and TXA 2 (Tbxa) prostanoid receptors via RT-qPCR; (2) the effect of iloprost, MRE-269, isoprenaline and ML277 on precontracted arterial tone in the presence of inhibitors of prostanoid receptors, potassium channels and the molecular interference of K V 7.1 via wire-myograph; (3) oestrus cycle stage via histological changes in cervical cell preparations., Key Results: Iloprost evoked a biphasic response in male mesenteric arteries, at concentrations ≤100 nmol·L -1 relaxing, then contracting the vessel at concentration ≥300 nmol·L -1 , a process attributed to IP and EP 3 receptors respectively. Secondary contraction was absent in the females, which was associated with a reduction in Ptger3. Pharmacological inhibition and molecular interference of K V 7.1 significantly attenuated relaxations produced by the selective IP receptor agonist MRE-269 in male and female Wistar in dioestrus/metoestrus, but not pro-oestrus/oestrus., Conclusions and Implications: Stark sexual dimorphisms in iloprost-mediated vasoactive responses are present within mesenteric arteries. K V 7.1 is implicated in IP receptor-mediated vasorelaxation and is impaired by the oestrus cycle., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Ca 2+ -activated Cl - channels (CaCCs) perform a multitude of functions including the control of cell excitability, regulation of cell volume and ionic homeostasis, exocrine and endocrine secretion, fertilization, amplification of olfactory sensory function, and control of smooth muscle cell contractility. CaCCs are the translated products of two members (ANO1 and ANO2, also known as TMEM16A and TMEM16B) of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca 2+ , post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca 2+ or indirectly through the intervention of the Ca 2+ -binding protein Calmodulin (CaM), and the structural domains responsible for Ca 2+ - and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl-(4,5)-bisphosphate (PIP 2 ), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ + channel β subunits KCNE1/5.

Hypertension is a major cause of cardiovascular morbidity and mortality, despite the availability of antihypertensive drugs with different targets and mechanisms of action. Here, we provide evidence that pharmacological inhibition of TMEM16A (ANO1), a calcium-activated chloride channel expressed in vascular smooth muscle cells, blocks calcium-activated chloride currents and contraction in vascular smooth muscle in vitro and decreases blood pressure in spontaneously hypertensive rats. The acylaminocycloalkylthiophene TMinh-23 fully inhibited calcium-activated TMEM16A chloride current with nanomolar potency in Fischer rat thyroid cells expressing TMEM16A, and in primary cultures of rat vascular smooth muscle cells. TMinh-23 reduced vasoconstriction caused by the thromboxane mimetic U46619 in mesenteric resistance arteries of wild-type and spontaneously hypertensive rats, with a greater inhibition in spontaneously hypertensive rats. Blood pressure measurements by tail-cuff and telemetry showed up to a 45-mmHg reduction in systolic blood pressure lasting for four-six hours in spontaneously hypertensive rats after a single dose of TMinh-23. A minimal effect on blood pressure was seen in wild-type rats or mice treated with TMinh-23. Five-day twice daily treatment of spontaneously hypertensive rats with TMinh-23 produced sustained reductions of 20-25 mmHg in daily mean systolic and diastolic blood pressure. TMinh-23 action was reversible, with blood pressure returning to baseline in spontaneously hypertensive rats by three days after treatment discontinuation. Thus, our studies provide validation for TMEM16A as a target for antihypertensive therapy and demonstrate the efficacy of TMinh-23 as an antihypertensive with a novel mechanism of action., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The dynein motor protein transports proteins away from the cell membrane along the microtubule network. Recently, we found the microtubule network was important for regulating the membrane abundance of voltage-gated Kv7.4 potassium channels in vascular smooth muscle. Here, we aimed to investigate the influence of dynein on the microtubule-dependent internalization of the Kv7.4 channel. Patch-clamp recordings from HEK293B cells showed Kv7.4 currents were increased after inhibiting dynein function with ciliobrevin D or by coexpressing p50/dynamitin, which specifically interferes with dynein motor function. Mutation of a dynein-binding site in the Kv7.4 C terminus increased the Kv7.4 current and prevented p50 interference. Structured illumination microscopy, proximity ligation assays, and coimmunoprecipitation showed colocalization of Kv7.4 and dynein in mesenteric artery myocytes. Ciliobrevin D enhanced mesenteric artery relaxation to activators of Kv7.2-Kv7.5 channels and increased membrane abundance of Kv7.4 protein in isolated smooth muscle cells and HEK293B cells. Ciliobrevin D failed to enhance the negligible S-1-mediated relaxations after morpholino-mediated knockdown of Kv7.4. Mass spectrometry revealed an interaction of dynein with caveolin-1, confirmed using proximity ligation and coimmunoprecipitation assays, which also provided evidence for interaction of caveolin-1 with Kv7.4, confirming that Kv7.4 channels are localized to caveolae in mesenteric artery myocytes. Lastly, cholesterol depletion reduced the interaction of Kv7.4 with caveolin-1 and dynein while increasing the overall membrane expression of Kv7.4, although it attenuated the Kv7.4 current in oocytes and interfered with the action of ciliobrevin D and channel activators in arterial segments. Overall, this study shows that dynein can traffic Kv7.4 channels in vascular smooth muscle in a mechanism dependent on cholesterol-rich caveolae., (© 2021 van der Horst et al.)

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded k V 7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of k V 7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of K V 7 channels within rat mesenteric artery ECs. Experimental Approach: In rat mesenteric artery, KCNQ transcript and K V 7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity. Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. K V 7.1, K V 7.4 and K V 7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different K V 7.2-5 activators S-1 and ML213. K IR 2 blockers ML133, and BaCl 2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. K V 7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusion and Implications: In rat mesenteric artery ECs, K V 7.4 and K V 7.5 channels are expressed, functionally interact with endothelial K IR 2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies K V 7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Baldwin, Sandow, Mondéjar-Parreño, Stott and Greenwood.)

Objective: The SMIT1 (sodium:myo-inositol transporter 1) regulates myo-inositol movement into cells and responses to hypertonic stimuli. Alteration of myo-inositol levels has been associated with several diseases, including hypertension, but there is no evidence of a functional role of SMIT1 in the vasculature. Recent evidence showed that in the nervous system SMIT1 interacted and modulated the function of members of the Kv7 family of voltage-gated potassium channels, which are also expressed in the vasculature where they regulate arterial contractility. Therefore, in this study, we evaluated whether SMIT1 was functionally relevant in arterial smooth muscle. Approach and Results: Immunofluorescence and polymerase chain reaction experiments revealed that SMIT1 was expressed in rat renal and mesenteric vascular smooth muscle cells. Isometric tension recordings showed that incubation of renal arteries with raffinose and myo-inositol (which increases SMIT1 expression) reduced the contractile responses to methoxamine, an effect that was abolished by preincubation with the pan-Kv7 blocker linopirdine and by molecular knockdown of Kv7.4 and Kv7.5. Knockdown of SMIT1 increased the contraction of renal arteries induced by methoxamine, impaired the response to the Kv7.2-Kv7.5 activator ML213 but did not interfere with the relaxant responses induced by openers of other potassium channels. Proximity ligation assay showed that SMIT1 interacted with heteromeric channels formed by Kv7.4 and Kv7.5 proteins in both renal and mesenteric vascular smooth muscle cells. Patch-clamp experiments showed that incubation with raffinose plus myo-inositol increased Kv7 currents in vascular smooth muscle cells., Conclusions: SMIT1 protein is expressed in vascular smooth muscle cells where it modulates arterial contractility through an association with Kv7.4/Kv7.5 heteromers.

K + channels play a fundamental role regulating membrane potential of pulmonary artery (PA) smooth muscle cells and their impairment is a common feature in pulmonary arterial hypertension (PAH). K + voltage-gated channel subfamily Q ( KCNQ1-5 ) or Kv7 channels and their regulatory subunits subfamily E (KCNE) regulatory subunits are known to regulate vascular tone, but whether Kv7 channel function is impaired in PAH and how this can affect the rationale for targeting Kv7 channels in PAH remains unknown. Here, we have studied the role of Kv7/KCNE subunits in rat PA and their possible alteration in PAH. Using the patch-clamp technique, we found that the total K + current is reduced in PA smooth muscle cells from pulmonary hypertension animals (SU5416 plus hypoxia) and Kv7 currents made a higher contribution to the net K + current. Likewise, enhanced vascular responses to Kv7 channel modulators were found in pulmonary hypertension rats. Accordingly, KCNE4 subunit was highly upregulated in lungs from pulmonary hypertension animals and patients. Additionally, Kv7 channel activity was enhanced in the presence of Kv1.5 and TASK-1 channel inhibitors and this was associated with an increased KCNE4 membrane abundance. Compared with systemic arteries, PA showed a poor response to Kv7 channel modulators which was associated with reduced expression and membrane abundance of Kv7.4 and KCNE4. Our data indicate that Kv7 channel function is preserved and KCNE4 is upregulated in PAH. Therefore, compared with other downregulated channels, the contribution of Kv7 channels is increased in PAH resulting in an enhanced sensitivity to Kv7 channel modulators. This study provides insight into the potential usefulness of targeting Kv7 channels in PAH.

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP 2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP 2 -binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca 2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α 1 -adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP 2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca 2+ currents, which were prevented by depleting PIP 2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP 2 into the local environment where it increases voltage-gated Ca 2+ channel activity., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Voltage-gated Kv7 potassium channels, encoded by KCNQ genes, have major physiological impacts cardiac myocytes, neurons, epithelial cells, and smooth muscle cells. Cyclic adenosine monophosphate (cAMP), a well-known intracellular secondary messenger, can activate numerous downstream effector proteins, generating downstream signaling pathways that regulate many functions in cells. A role for cAMP in ion channel regulation has been established, and recent findings show that cAMP signaling plays a role in Kv7 channel regulation. Although cAMP signaling is recognized to regulate Kv7 channels, the precise molecular mechanism behind the cAMP-dependent regulation of Kv7 channels is complex. This review will summarize recent research findings that support the mechanisms of cAMP-dependent regulation of Kv7 channels., (Copyright © 2020 van der Horst, Greenwood and Jepps.)

Within the vasculature Kv7 channels are key regulators of basal tone and contribute to a variety of receptor mediated vasorelaxants. The Kv7.4 isoform, abundant within the vasculature, is key to these processes and was recently shown to have an obligatory requirement of G-protein βγ subunits for its voltage dependent activity. There is an increasing appreciation that with 5 Gβ subunits and 12 Gγ subunits described in mammalian cells that different Gβ x γ x combinations can confer selectivity in Gβγ effector stimulation. Therefore, we aimed to characterize the Gβ subunit(s) which basally regulate Kv7.4 channels and native vascular Kv7 channels. In Chinese Hamster Ovary cells overexpressing Kv7.4 and different Gβx subunits only Gβ1, Gβ3, and Gβ5 enhanced Kv7.4 currents, increasing the activation kinetics and negatively shifting the voltage dependence of activation. In isolated rat renal artery myocytes, proximity ligation assay detected an interaction of Kv7.4 with Gβ1 and Gβ3 subunits, but not other isoforms. Morpholino directed knockdown of Gβ1 in rat renal arteries did not alter Kv7 dependent currents but reduced Kv7.4 protein expression. Knockdown of Gβ3 in rat renal arteries resulted in decreased basal K + currents which were not sensitive to pharmacological inhibition of Kv7 channels. These studies implicate the Gβ1 subunit in the synthesis or stability of Kv7.4 proteins, whilst revealing that the Gβ3 isoform is responsible for the basal activity of Kv7 channels in native rat renal myocytes. These findings demonstrate that different Gβ subunits have important individual roles in ion channel regulation., (Copyright © 2020 Greenwood and Stott.)

This study explored the mechanism by which Ca 2+ -activated Cl - channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca 2+ -activated Cl - currents ( I ClCa ) were recorded from HEK-293 cells expressing mouse TMEM16A. I ClCa were evoked using a pipette solution in which free Ca 2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, I ClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. I ClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. I ClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of I ClCa by CaMKII.

Background and Purpose: Coronary artery disease leads to ischaemic heart disease and ultimately myocardial infarction. Thus, it is important to determine the factors that regulate coronary blood flow. Ca 2+ -activated chloride channels contribute to the regulation of arterial tone; however, their role in coronary arteries is unknown. The aim of this study was to investigate the expression and function of the main molecular correlate of Ca 2+ -activated chloride channels, TMEM16A, in rat coronary arteries., Experimental Approach: We performed mRNA and protein analysis, electrophysiological studies of coronary artery myocytes, and functional studies of coronary artery contractility and coronary perfusion, using novel inhibitors of TMEM16A. Furthermore, we assessed whether any changes in expression and function occurred in coronary arteries from spontaneously hypertensive rats (SHRs)., Key Results: TMEM16A was expressed in rat coronary arteries. The TMEM16A-specific inhibitor, MONNA, hyperpolarised the membrane potential in U46619. MONNA, T16A inh -A01, and Ani9 attenuated 5-HT/U46619-induced contractions. MONNA and T16A inh -A01 also increased coronary flow in Langendorff perfused rat heart preparations. TMEM16A mRNA was increased in coronary artery smooth muscle cells from SHRs, and U46619 and 5-HT were more potent in arteries from SHRs than in those from normal Wistar rats. MONNA diminished this increased sensitivity to U46619 and 5-HT., Conclusions and Implications: In conclusion, TMEM16A is a key regulator of coronary blood flow and is implicated in the altered contractility of coronary arteries from SHRs., (© 2019 The British Pharmacological Society.)

Objective- In renal arteries, inhibitors of G protein βγ subunits (Gβγ) reduce Kv7 activity and inhibit Kv7-dependent receptor-mediated vasorelaxations. However, the mechanisms underlying receptor-mediated relaxation are artery specific. Consequently, the aim of this study was to ascertain the role of Gβγ in Kv7-dependent vasorelaxations of the rat vasculature. Approach and Results- Isometric tension recording was performed in isolated rat renal, mesenteric, and cerebral arteries to study isoproterenol and calcitonin gene-related peptide relaxations. Kv7.4 was knocked down via morpholino transfection while inhibition of Gβγ was investigated with gallein and M119K. Proximity ligation assay was performed on isolated myocytes to study the association between Kv7.4 and G protein β subunits or signaling intermediaries. Isoproterenol or calcitonin gene-related peptide-induced relaxations were attenuated by Kv7.4 knockdown in all arteries studied. Inhibition of Gβγ with gallein or M119K had no effect on isoproterenol-mediated relaxations in mesenteric artery but had a marked effect on calcitonin gene-related peptide-induced responses in mesenteric artery and cerebral artery and isoproterenol responses in renal artery. Isoproterenol increased association with Kv7.4 and Rap1a in mesenteric artery which were not sensitive to gallein, whereas in renal artery, isoproterenol increased Kv7.4-AKAP (A-kinase anchoring protein) associations in a gallein-sensitive manner. Conclusions- The Gβγ-Kv7 relationship differs between vessels and is an essential requirement for AKAP, but not Rap-mediated regulation of the channel.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Voltage-gated K v 7.4 channels have been implicated in vascular smooth muscle cells' activity because they modulate basal arterial contractility, mediate responses to endogenous vasorelaxants, and are downregulated in several arterial beds in different models of hypertension. Angiotensin II (Ang II) is a key player in hypertension that affects the expression of several classes of ion channels. In this study, we evaluated the effects of Ang II on the expression and function of vascular K v 7.4. Western blot and quantitative polymerase chain reaction revealed that in whole rat mesenteric artery, Ang II incubation for 1 to 7 hours decreased K v 7.4 protein expression without reducing transcript levels. Moreover, Ang II decreased XE991 (K v 7)-sensitive currents and attenuated membrane potential hyperpolarization and relaxation induced by the K v 7 activator ML213. Ang II also reduced K v 7.4 staining at the plasma membrane of vascular smooth muscle cells. Proteasome inhibition with MG132 prevented Ang II-induced decrease of K v 7.4 levels and counteracted the functional impairment of ML213-induced relaxation in myography experiments. Proximity ligation assays showed that Ang II impaired the interaction of K v 7.4 with the molecular chaperone HSP90 (heat shock protein 90), enhanced the interaction of K v 7.4 with the E3 ubiquitin ligase CHIP (C terminus of Hsp70-interacting protein), and increased K v 7.4 ubiquitination. Similar alterations were found in mesenteric vascular smooth muscle cells isolated from Ang II-infused mice. The effect of Ang II was emulated by 17-AAG (17-demethoxy-17-(2-propenylamino) geldanamycin) that inhibits HSP90 interactions with client proteins. These results show that Ang II downregulates K v 7.4 by altering protein stability through a decrease of its interaction with HSP90. This leads to the recruitment of CHIP and K v 7.4 ubiquitination and degradation via the proteasome., (© 2018 American Heart Association, Inc.)

K v 7 channels are voltage-gated potassium channels encoded by KCNQ genes that have a considerable physiological impact in many cell types. This reliance upon K v 7 channels for normal cellular function, as well as the existence of hereditary disorders caused by mutations to KCNQ genes, means that pharmacological targeting of these channels has broad appeal. Consequently, a plethora of chemical entities that modulate K v 7 channel activity have been developed. Moreover, K v 7 channels are influenced by many disparate intracellular mediators and trafficking processes, making upstream targeting an appealing prospect for therapeutic development. This review covers the main characteristics of these multifunctional and versatile channels with the aim of providing insight into the therapeutic value of targeting these channels.

K + channels encoded by the ether-a-go-go related gene (ERG1 or KCNH2) are important determinants of the cardiac action potential. Expression of both cardiac isoforms (ERG1a and ERG1b) were identified in murine portal vein and distinctive voltage-gated K + currents were recorded from single myocytes. The aim of the present study was to ascertain the expression and functional impact of ERG channels in murine arteries. Methods: Quantitative RT-PCR was undertaken on RNA extracted from a number of murine arteries. Immunofluorescence was performed on single vascular smooth muscle cells using antibodies against the ERG1 expression product (Kv11.1). Single cell electrophysiology was performed on myocytes from portal vein and several different arteries, complimented by isometric tension recordings. Proliferation assays were undertaken on smooth muscle cells isolated from femoral arteries. Results: ERG1 transcripts were detected in all murine blood vessels, and Kv11.1 immunofluorescence was observed in all smooth muscle cells. However, K + currents with properties consistent with ERG channels were only recorded in portal vein myocytes. Moreover, ERG channel blockers (E4031 or dofetilide, 1 μM) failed to depolarize carotid arteries or produce contraction. Proliferation of arterial smooth muscle cells was associated with a marked increase in ERG1 expression and ERG blockers suppressed proliferation significantly. Conclusions: These data reveal that arterial blood vessels express ERG channels that appear to be functional silent in contractile smooth muscle but contribute to proliferative response.

Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP 2 ). βγ G proteins (Gβγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gβγ increased Kv7.4 open probability through an increased sensitivity to PIP 2 . In HEK cells stably expressing Kv7.4, PIP 2 or Gβγ increased open probability in a concentration dependent manner. Depleting PIP 2 prevented any Gβγ-mediated stimulation whilst an array of Gβγ inhibitors prohibited any PIP 2 -induced current enhancement. A combination of PIP 2 and Gβγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP 2 depletion or Gβγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gβγ., Competing Interests: The authors declare that they have no conflicts of interest.

Objective: To establish the role of Kv7 channels in EPAC (exchange protein directly activated by cAMP)-dependent relaxations of the rat vasculature and to investigate whether this contributes to β-adrenoceptor-mediated vasorelaxations., Approach and Results: Isolated rat renal and mesenteric arteries (RA and MA, respectively) were used for isometric tension recording to study the relaxant effects of a specific EPAC activator and the β-adrenoceptor agonist isoproterenol in the presence of potassium channel inhibitors and cell signaling modulators. Isolated myocytes were used in proximity ligation assay studies to detect localization of signaling intermediaries with Kv7.4 before and after cell stimulation. Our studies showed that the EPAC activator (8-pCPT-2Me-cAMP-AM) produced relaxations and enhanced currents of MA and RA that were sensitive to linopirdine (Kv7 inhibitor). Linopirdine also inhibited isoproterenol-mediated relaxations in both RA and MA. In the MA, isoproterenol relaxations were sensitive to EPAC inhibition, but not protein kinase A inhibition. In contrast, isoproterenol relaxations in RA were attenuated by protein kinase A but not by EPAC inhibition. Proximity ligation assay showed a localization of Kv7.4 with A-kinase anchoring protein in both vessels in the basal state, which increased only in the RA with isoproterenol stimulation. In the MA, but not the RA, a localization of Kv7.4 with both Rap1a and Rap2 (downstream of EPAC) increased with isoproterenol stimulation., Conclusions: EPAC-dependent vasorelaxations occur in part via activation of Kv7 channels. This contributes to the isoproterenol-mediated relaxation in mesenteric, but not renal, arteries., (© 2016 American Heart Association, Inc.)

Aims: Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischaemic cardiac injury of Kv7.4 channels have been investigated., Methods and Results: Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by quantitative polymerase chain reaction. Western blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with ∼ 30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immunogold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 µM) and flupirtine (30 µM), two selective Kv7 activators, increased Tl(+) influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca(2+) levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/re-oxygenation and largely prevented the functional and morphological changes triggered by global ischaemia/reperfusion (I/R) in Langendorff-perfused rat hearts., Conclusion: Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Background and Purpose: KCNQ-encoded voltage-dependent potassium channels (Kv 7) are involved in the regulation of vascular tone. In this study we evaluated the influence of Kv 7 channel activation on smooth muscle relaxation in rat penile arteries and corpus cavernosum from normal and spontaneously hypertensive, heart failure-prone (SHHF) rats - a rat model of human metabolic syndrome., Experimental Approach: Quantitative PCR and immunohistochemistry were used to determine the expression of KCNQ isoforms in penile tissue. Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from normal and SHHF rats., Key Results: Transcripts for KCNQ3, KCNQ4 and KCNQ5 were detected in penile arteries and corpus cavernosum. KCNQ1 was only found in corpus cavernosum. Immunofluorescence signals to Kv 7.4 and Kv 7.5 were found in penile arteries, penile veins and corpus cavernosum. The Kv 7.2-7.5 activators, ML213 and BMS204352, relaxed pre-contracted penile arteries and corpus cavernosum independently of nitric oxide synthase or endothelium-derived hyperpolarization. Relaxations to sildenafil, a PDE5 inhibitor, and sodium nitroprusside (SNP), an nitric oxide donor, were reduced by blocking Kv 7 channels with linopirdine in penile arteries and corpus cavernosum. In SHHF rat penile arteries and corpus cavernosum, relaxations to ML213 and BMS204352 were attenuated, and the blocking effect of linopirdine on sildenafil-induced and SNP-induced relaxations reduced. KCNQ3, KCNQ4 and KCNQ5 were down-regulated, and KCNQ1 was up-regulated in corpus cavernosum from SHHF rats. KCNQ1-5 transcripts remained unchanged in penile arteries from SHHF rats., Conclusions and Implications: These data suggest that Kv 7 channels play a role in erectile function and contribute to the pathophysiology of erectile dysfunction, an early indicator of cardiovascular disease., (© 2016 The British Pharmacological Society.)

Key Points: KCNE4 alters the biophysical properties and cellular localization of voltage-gated potassium channel Kv7.4. KCNE4 is expressed in a variety of arteries and, in mesenteric arteries, co-localizes with Kv7.4, which is important in the control of vascular contractility. Knockdown of KCNE4 leads to reduced Kv7.4 membrane abundance, a depolarized membrane potential and an augmented response to vasoconstrictors. KCNE4 is a key regulator of the function and expression of Kv7.4 in vascular smooth muscle., Abstract: The KCNE ancillary subunits (KCNE1-5) significantly alter the expression and function of voltage-gated potassium channels; however, their role in the vasculature has yet to be determined. The present study aimed to investigate the expression and function of the KCNE4 subunit in rat mesenteric arteries and to determine whether it has a functional impact on the regulation of arterial tone by Kv7 channels. In HEK cells expressing Kv7.4, co-expression of KCNE4 increased the membrane expression of Kv7.4 and significantly altered Kv7.4 current properties. Quantitative PCR analysis of different rat arteries found that the KCNE4 isoform predominated and proximity ligation experiments showed that KCNE4 co-localized with Kv7.4 in mesenteric artery myocytes. Morpholino-induced knockdown of KCNE4 depolarized mesenteric artery smooth muscle cells and resulted in their increased sensitivity to methoxamine being attenuated (mean ± SEM EC50 decreased from 5.7 ± 0.63 μm to 1.6 ± 0.23 μm), which coincided with impaired effects of Kv7 modulators. When KCNE4 expression was reduced, less Kv7.4 expression was found in the membrane of the mesenteric artery myocytes. These data show that KCNE4 is consistently expressed in a variety of arteries, and knockdown of the expression product leads to reduced Kv7.4 membrane abundance, a depolarized membrane potential and an augmented response to vasoconstrictors. The present study is the first to demonstrate an integral role of KCNE4 in regulating the function and expression of Kv7.4 in vascular smooth muscle., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Ischemic stroke can cause striatal dopamine efflux that contributes to cell death. Since Kv7 potassium channels regulate dopamine release, we investigated the effects of their pharmacological modulation on dopamine efflux, measured by fast cyclic voltammetry (FCV), and neurotoxicity, in Wistar rat caudate brain slices undergoing oxygen and glucose deprivation (OGD). The Kv7 activators retigabine and ICA27243 delayed the onset, and decreased the peak level of dopamine efflux induced by OGD; and also decreased OGD-induced damage measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Retigabine also reduced OGD-induced necrotic cell death evaluated by lactate dehydrogenase activity assay. The Kv7 blocker linopirdine increased OGD-evoked dopamine efflux and OGD-induced damage, and attenuated the effects of retigabine. Quantitative-PCR experiments showed that OGD caused an ~6-fold decrease in Kv7.2 transcript, while levels of mRNAs encoding for other Kv7 subunits were unaffected; western blot experiments showed a parallel reduction in Kv7.2 protein levels. Retigabine also decreased the peak level of dopamine efflux induced by L-glutamate, and attenuated the loss of TTC staining induced by the excitotoxin. These results suggest a role for Kv7.2 in modulating ischemia-evoked caudate damage.