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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.

BACKGROUND AND PURPOSEKCNQ-encoded voltage-gated potassium channels (Kv7) have recently been identified as important anti-constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo. EXPERIMENTAL APPROACH Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show Kv7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels. KEY RESULTSKCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. Kv7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the Kv7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K+ currents. In contrast, the Kv7.1-specific blocker chromanol 293B did not affect vascular tone. Two Kv7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. Kv7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991. CONCLUSIONS AND IMPLICATIONS This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo. LINKED ARTICLE This article is commented on by Mani and Byron, pp. 38–41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x

In isolated human atrial cardiomyocytes, inhibition of K2P3.1 K(+) channels results in action potential (action potential duration (APD)) prolongation. It has therefore been postulated that K2P3.1 (KCNK3), together with K2P9.1 (KCNK9), could represent novel drug targets for the treatment of atrial fibrillation (AF). However, it is unknown whether these findings in isolated cells translate to the whole heart. The purposes of this study were to investigate the expression levels of KCNK3 and KCNK9 in human hearts and two relevant rodent models and determine the antiarrhythmic potential of K2P3.1 inhibition in isolated whole-heart preparations. By quantitative PCR, we found that KCNK3 is predominantly expressed in human atria whereas KCNK9 was not detectable in heart human tissue. No differences were found between patients in AF or sinus rhythm. The expression in guinea pig heart resembled humans whereas rats displayed a more uniform expression of KCNK3 between atria and ventricle. In voltage-clamp experiments, ML365 and A293 were found to be potent and selective inhibitors of K2P3.1, but at pH 7.4, they failed to prolong atrial APD and refractory period (effective refractory period (ERP)) in isolated perfused rat and guinea pig hearts. At pH 7.8, which augments K2P3.1 currents, pharmacological channel inhibition produced a significant prolongation of atrial ERP (11.6 %, p = 0.004) without prolonging ventricular APD but did not display a significant antiarrhythmic effect in our guinea pig AF model (3/8 hearts converted on A293 vs 0/7 hearts in time-matched controls). These results suggest that when K2P3.1 current is augmented, K2P3.1 inhibition leads to atrial-specific prolongation of ERP; however, this ERP prolongation did not translate into significant antiarrhythmic effects in our AF model.

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.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.

The Kv7 family of voltage-gated potassium channels are expressed within the vasculature where they are key regulators of vascular tone and mediate cAMP-linked endogenous vasodilator responses, a pathway that is compromised in hypertension. However, the role of Kv7 channels in non–cAMP-linked vasodilator pathways has not been investigated. Natriuretic peptides are potent vasodilators, which operate primarily through the activation of a cGMP-dependent signaling pathway. This study investigated the putative role of Kv7 channels in natriuretic peptide–dependent relaxations in the vasculature of normal and hypertensive animals. Relaxant responses of rat aorta to both atrial and C-type natriuretic peptides and the nitric oxide donor sodium nitroprusside were impaired by the Kv7 blocker linopirdine (10 μmol/L) but not by the Kv7.1-specific blocker HMR1556 (10 μmol/L) and other K + channel blockers. In contrast, only the atrial natriuretic peptide response was sensitive to linopirdine in the renal artery. These Kv7-mediated responses were attenuated in arteries from hypertensive rats. Quantitative polymerase chain reaction showed that A- and B-type natriuretic peptide receptors were expressed at high levels in the aorta and renal artery from normal and spontaneously hypertensive rats. This study provides the first evidence that natriuretic peptide responses are impaired in hypertension and that recruitment of Kv7 channels is a key component of natriuretic peptide–dependent vasodilations.

Objective— Middle cerebral artery (MCA) diameter is regulated by inherent myogenic activity and the effect of potent vasodilators such as calcitonin gene-related peptide (CGRP). Previous studies showed that MCAs express KCNQ1, 4, and 5 potassium channel genes, and the expression products (Kv7 channels) participate in the myogenic control of MCA diameter. The present study investigated the contribution of Kv7.4 and Kv7.5 isoforms to myogenic and CGRP regulation of MCA diameter and determined whether they were affected in hypertensive animals. Approach and Results— Isometric tension recordings performed on MCA from normotensive rats produced CGRP vasodilations that were inhibited by the pan-Kv7 channel blocker linopirdine ( P P Conclusions— This study reveals, for the first time to our knowledge, that in cerebral arteries, Kv7.4 and Kv7.5 proteins exist predominantly as a functional heterotetramer, which regulates intrinsic myogenicity and vasodilation attributed to CGRP. Surprisingly, unlike systemic arteries, Kv7 activity in MCAs is not affected by the development of hypertension, and CGRP-mediated vasodilation is well maintained. As such, cerebrovascular Kv7 channels could be amenable for therapeutic targeting in conditions such as cerebral vasospasm.

The goal of the present study was to determine the role of KCNQ -encoded K v channels (K v 7 channels) in the passive and active regulation of coronary flow in normotensive and hypertensive rats. In left anterior descending coronary arteries from normotensive rats, structurally different K v 7.2 to 7.5 activators produced relaxations, which were considerably less in arteries from hypertensive rats and were not mimicked by the K v 7.1-specific activator R-L3. In isolated, perfused heart preparations, coronary flow rate increased in response to the K v 7.2 to 7.5 activator (S)-1 and was diminished in the presence of a K v 7 inhibitor. The expression levels of KCNQ1–5 and their known accessory KCNE1–5 subunits in coronary arteries were similar in normotensive and hypertensive rats as measured by quantitative polymerase chain reaction. However, K v 7.4 protein expression was reduced in hypertensive rats. Application of adenosine or A2A receptor agonist CGS-21680 produced concentration-dependent relaxations of coronary arteries from normotensive rats, which were attenuated by application of K v 7 inhibitors. K v 7 blockers also attenuated the ischemia-induced increase in coronary perfusion in Langendorff studies. Overall, these data establish K v 7 channels as crucial regulators of coronary flow at resting and after hypoxic insult.

Potassium channels are key regulators of smooth muscle tone, with increases in activity resulting in hyperpolarisation of the cell membrane, which acts to oppose vasoconstriction. Several potassium channels exist within smooth muscle, but the KV7 family of voltage-gated potassium channels have been identified as being crucial mediators of this process in a variety of smooth muscle. Recently, KV7 channels have been shown to be involved in the pathogenesis of hypertension, as well as being implicated in other smooth muscle disorders, providing a new and inviting target for smooth muscle disorders.

Background— Voltage-gated potassium (K + ) channels encoded by KCNQ genes (Kv7 channels) have been identified in various rodent and human blood vessels as key regulators of vascular tone; however, nothing is known about the functional impact of these channels in vascular disease. We ascertained the effect of 3 structurally different activators of Kv7.2 through Kv7.5 channels (BMS-204352, S-1, and retigabine) on blood vessels from normotensive and hypertensive animals. Methods and Results— Precontracted thoracic aorta and mesenteric artery segments from normotensive rats were relaxed by all 3 Kv7 activators, with potencies of BMS-204352=S-1>retigabine. We also tested these agents in the coronary circulation using the Langendorff heart preparation. BMS-204352 and S-1 dose dependently increased coronary perfusion at concentrations between 0.1 and 10 μmol/L, whereas retigabine was effective at 1 to 10 μmol/L. In addition, S-1 increased K + currents in isolated mesenteric artery myocytes. The ability of these agents to relax precontracted vessels, increase coronary flow, or augment K + currents was impaired considerably in tissues isolated from spontaneously hypertensive rats (SHRs). Of the 5 KCNQ genes, only the expression of KCNQ4 was reduced (≈3.7 fold) in SHRs aorta. Kv7.4 protein levels were ≈50% lower in aortas and mesenteric arteries from spontaneously hypertensive rats compared with normotensive vessels. A similar attenuated response to S-1 and decreased Kv7.4 were observed in mesenteric arteries from mice made hypertensive by angiotensin II infusion compared with normotensive controls. Conclusions— In 2 different rat and mouse models of hypertension, the functional impact of Kv7 channels was dramatically downregulated.

Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca2+-activated Cl− currents ( IClCa) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl− channels are a major depolarizing mechanism. Qualitatively similar Cl− currents were evoked by a pipette solution containing 500 nM Ca2+ in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle. Western blot analysis with different antibodies directed against TMEM16A revealed a number of products with a consistent band at ∼120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-μm-thick slices of portal vein, thoracic aorta, and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an ∼147-kDa mouse TMEM16A-green fluorescent protein (GFP) fusion protein expressed in HEK 293 cells, which correlated to a similar band detected by a GFP antibody. Patch-clamp experiments revealed that IClCa generated by transfection of TMEM16A-GFP in HEK 293 cells displayed remarkable similarities to IClCa recorded in vascular myocytes, including slow kinetics, steep outward rectification, and a response similar to the pharmacological agent niflumic acid. This study shows that TMEM16A expression is robust in murine vascular smooth muscle cells, consolidating the view that this gene is a viable candidate for the native Ca2+-activated Cl− channel in this cell type.

Members of the Kv7 voltage-gated K+ channel family are important determinants of cardiac and neuronal membrane excitability. Recently, we and others have shown that Kv7 channels are also crucial regulators of smooth muscle activity. The aim of the present study was to assess the Kv7 expression in different parts of the murine gastrointestinal (GI) tract and to assess their functional roles by use of pharmacological agents. Of KCNQ/Kv7 members, both KCNQ4/Kv7.4 and KCNQ5/Kv7.5 genes and proteins were the most abundantly expressed Kv7 channels in smooth muscles throughout the GI tract. Immunohistochemical staining also revealed that Kv7.4 and Kv7.5 but not Kv7.1 were expressed in the circular muscle layer of the colon. In segments of distal colon circular muscle exhibiting spontaneous phasic contractions, the nonselective Kv7 blockers XE991 and linopirdine increased the integral of tension. Increases in the integral of tension were also observed under conditions of neuronal blockade. Similar effects, although less marked, were observed in the proximal colon. As expected, the Kv7.1-selective blocker chromanol 293B had no effect in either type of segment. These data show that Kv7.x especially Kv7.4 and Kv7.5 are expressed in different regions of the murine gastrointestinal tract and blockers of Kv7 channels augment inherent contractile activity. Drugs that selectively block Kv7.4/7.5 might be promising therapeutics for the treatment of motility disorders such as constipation associated with irritable bowel syndrome.

Calcium activated chloride (ClCa) channels are an important contractile mechanism in smooth muscle cells. Activation of these channels by calcium (Ca2+) ions leads to Cl- efflux and membrane depolarization. This depolarization then favors the activation of voltage-gated Ca2+ channels (e.g. L-type), providing a positive feed-back loop that allows for sustained contraction.The molecular candidate encoding for this channel has remained elusive, although a number of suggestions have been investigated. The latest contender to emerge is TMEM16A (Anoctamin). Evidence so far confirms that over-expression of TMEM16A generates Cl- currents with the same time-dependent kinetics and Ca2+ sensitivity as recorded through ClCa channels in smooth muscle cells.This study investigated expression profiles of TMEM16A in a number of vascular tissues across a number of species. The aim of this study was to confirm the role of TMEM16A as a potential candidate encoding for ClCa currents (IClCa) in native vascular myocytes.Smooth muscle cells were enzymatically isolated from the mouse portal vein (PV), aorta and carotid artery (CA), rat pulmonary artery (PA), and the rabbit PA. Robust IClCa, exhibiting distinctive voltage-dependent kinetics, were recorded from all of the tissue preparations mentioned.RT-PCR was performed, using species-specific primers designed against the available sequences for TMEM16A. Mouse aorta, PA, CA and PV, rat aorta, PA and PV, and rabbit PA tissues were studies. All samples showed clear expression of TMEM16A.Immunocytochemistry revealed specific expression of TMEM16A in rat PA, and mouse PV, CA and aorta isolated myocytes. This was apparent throughout the cytoplasm, with punctuate “hotspots”. Western blot analysis showed bands of the expected molecular weight in mouse aorta, PV and CA.In summary, we have demonstrated that TMEM16A is a viable candidate for the encoding of ClCa channels in vascular smooth muscle.