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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 KV. Vascular potassium channels, including KV, 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 KV 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.

Hypertension is associated with presence of vascular abnormalities, such as remodeling and rarefaction. These processes play an important role in cerebrovascular disease development, however, the mechanistic changes leading to these diseases are not well characterized. Using data-independent acquisition-based mass spectrometry analysis, we determined the protein changes in cerebral arteries in pre- and early-onset hypertension from the spontaneously hypertensive rat (SHR), a model that resembles essential hypertension. Our analysis identified 125 proteins with expression levels that were significantly up- or downregulated in 12-week old SHRs compared to normotensive Wistar Kyoto rats. Using an angiogenesis enrichment analysis, we identified a critical imbalance in angiogenic proteins, promoting an anti-angiogenic profile in cerebral arteries at the early-onset of hypertension. In a comparison to previously published data, we demonstrate that this angiogenic imbalance is not present in mesenteric and renal arteries from age-matched SHRs. Finally, we identified two proteins (Fbln5 and Cdh13), whose expression levels were critically altered in cerebral arteries compared to the other arterial beds. The observation of an angiogenic imbalance in cerebral arteries from the SHR reveals critical protein changes in the cerebrovasculature at the early-onset of hypertension and provides novel insight into the early pathology of cerebrovascular disease.

Aims: The aim of this study is to examine whether colchicine improves β adrenoceptor-mediated vasodilation in humans by conducting a double-blinded, placebo-controlled intervention study. Colchicine treatment has known beneficial effects on cardiovascular health and reduces the incidence of cardiovascular disease. Studies in isolated rodent arteries have shown that colchicine can enhance β adrenoceptor-mediated vasodilation, but this has not been determined in humans. Methods: Middle-aged men with essential hypertension were randomly assigned firstly to acute treatment with either 0.5 mg colchicine (n = 19) or placebo (n = 12). They were subsequently re-randomized for 3 weeks of treatment with either colchicine 0.5 mg twice daily (n = 16) or placebo (n = 15) followed by a washout period of 48–72 h. The vasodilator responses to isoprenaline, acetylcholine and sodium nitroprusside were determined as well as arterial pressure, arterial compliance and plasma inflammatory markers. Results: Acute colchicine treatment increased isoprenaline (by 38% for the highest dose) as well as sodium nitroprusside (by 29% main effect) -induced vasodilation but had no effect on the response to acetylcholine. The 3-week colchicine treatment followed by a washout period did not induce an accumulated or sustained effect on the β adrenoceptor response, and there was no effect on arterial pressure, arterial compliance or the level of measured inflammatory markers. Conclusion: Colchicine acutely enhances β adrenoceptor- and nitric oxide-mediated changes in vascular conductance in humans, supporting that the mechanism previously demonstrated in rodents, translates to humans. The results provide novel translational evidence for a transient enhancing effect of colchicine on β adrenoceptor-mediated vasodilation in humans with essential hypertension. Condensed abstract: Preclinical studies in isolated rodent arteries have shown that colchicine can enhance β adrenoceptor-mediated vasodilation. Here we show that this effect of colchicine can be translated to humans. Acute colchicine treatment was found to increase both isoprenaline- and sodium nitroprusside-induced vasodilation. The study provides the first translational evidence for a transient β adrenoceptor-mediated vasodilatory effect of colchicine in humans. The finding of an acute effect suggests that it may be clinically important to maintain an adequate bioavailability of colchicine.

Tree and shrub barks have been used as folk medicine by numerous cultures across the globe for millennia, for a variety of indications, including as vasorelaxants and antispasmodics. Here, using electrophysiology and myography, we discovered that the KCNQ5 voltage-gated potassium channel mediates vascular smooth muscle relaxant effects of barks used in Native American folk medicine. Bark extracts (1%) from Birch, Cramp Bark, Slippery Elm, White Oak, Red Willow, White Willow, and Wild Cherry each strongly activated KCNQ5 expressed in Xenopus oocytes. Testing of a subset including both the most and the least efficacious extracts revealed that Red Willow, White Willow, and White Oak KCNQ-dependently relaxed rat mesenteric arteries; in contrast, Black Haw bark neither activated KCNQ5 nor induced vasorelaxation. Two compounds common to the active barks (gallic acid and tannic acid) had similarly potent and efficacious effects on both KCNQ5 activation and vascular relaxation, and this together with KCNQ5 modulation by other tannins provides a molecular basis for smooth muscle relaxation effects of Native American folk medicine bark extracts.

BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation.METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone.RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries.CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.

Objective: Intravenous acetaminophen/paracetamol (APAP) is well documented to cause hypotension. Since the patients receiving intravenous APAP are usually critically ill, any severe hemodynamic changes, as with those associated with APAP, can be life-threatening. The mechanism underlying this dangerous iatrogenic effect of APAP was unknown. Approach and Results: Here, we show that intravenous APAP caused transient hypotension in rats, which was attenuated by the Kv7 channel blocker, linopirdine. APAP metabolite N-acetyl-p-benzoquinone imine caused vasodilatation of rat mesenteric arteries ex vivo. This vasodilatation was sensitive to linopirdine and also the calcitonin gene-related peptide antagonist, BIBN 4096. Further investigation revealed N-acetyl-p-benzoquinone imine stimulates calcitonin gene-related peptide release from perivascular nerves, causing a cAMP-dependent activation of Kv7 channels. We also show that N-acetyl-p-benzoquinone imine enhances Kv7.4 and Kv7.5 channels overexpressed in oocytes, suggesting that it can activate Kv7.4 and Kv7.5 channels directly, to elicit vasodilatation. Conclusions: Direct and indirect activation of Kv7 channels by the APAP metabolite N-acetyl-p-benzoquinone imine decreases arterial tone, which can lead to a drop in blood pressure. Our findings provide a molecular mechanism and potential preventive intervention for the clinical phenomenon of intravenous APAP-dependent transient hypotension.

Background: The voltage-gated potassium channel (Kv)7.4 and Kv7.5 channels contribute to the β-adrenoceptor-mediated vasodilatation. In arteries from hypertensive rodents, the Kv7.4 channel is downregulated and function attenuated, which contributes to the reduced β-adrenoceptor-mediated vasodilatation observed in these arteries. Recently, we showed that disruption of the microtubule network, with colchicine, or inhibition of the microtubule motor protein, dynein, with ciliobrevin D, enhanced the membrane abundance and function of Kv7.4 channels in rat mesenteric arteries. This study aimed to determine whether these pharmacological compounds can improve Kv7.4 function in third-order mesenteric arteries from the spontaneously hypertensive rat, thereby restoring the β-adrenoceptor-mediated vasodilatation. methods: Wire and intravital myography was performed on normotensive and hypertensive male rat mesenteric arteries and immunostaining was performed on isolated smooth muscle cells from the same arteries. Results: Using wire and intravital microscopy, we show that ciliobrevin D enhanced the β-adrenoceptor-mediated vasodilatation by isoprenaline. This effect was inhibited partially by the Kv7 channel blocker linopirdine and was dependent on an increased functional contribution of the β2-adrenoceptor to the isoprenaline-mediated relaxation. In mesenteric arteries from the spontaneously hypertensive rat, ciliobrevin D and colchicine both improved the isoprenaline-mediated vasorelaxation and relaxation to the Kv7.2 -7.5 activator, ML213. Immunostaining confirmed ciliobrevin D enhanced the membrane abundance of Kv7.4. As well as an increase in the function of Kv7.4, the functional changes were associated with an increase in the contribution of β2-adrenoceptor following isoprenaline treatment. Immunostaining experiments showed ciliobrevin D prevented isoprenaline-mediated internalizationof the β2-adrenoceptor. Conclusions: Overall, these data show that colchicine and ciliobrevin D can induce a β2-adrenoceptor-mediated vasodilatation in arteries from the spontaneously hypertensive rat as well as reinstating Kv7.4 channel function.

Activation of antiapoptotic signaling cascades, such as the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways, is protective in a variety of tissues in the context of ischemia-reperfusion (IR) injury. Hepatic IR injury causes clinically significant hepatocellular damage in surgical procedures, including liver transplantation and hepatic resection, increasing associated morbidity and mortality. We previously found that the cardiovascular-expressed K+ voltage-gated channel ancillary subunit KCNE4 sex specifically influences the cardiac RISK/SAFE pathway response to IR and that Kcne4 deletion testosterone dependently exacerbates cardiac IR injury. Here, we discovered that germline Kcne4 deletion exacerbates hepatic IR injury damage in 13-mo-old male mice, despite a lack of Kcne4 expression in male mouse liver. Examining RISK/SAFE pathway induction, we found that Kcne4 deletion prevents the hepatic ERK1/2 phosphorylation response to IR injury. Conversely, in 13-mo-old female mice, Kcne4 deletion increased both baseline and post-IR GSK-3β inhibitory phosphorylation, and pharmacological GSK-3β inhibition was hepatoprotective. Finally, castration of male mice restored normal hepatic RISK/SAFE pathway responses in Kcne4−/− mice, eliminated Kcne4 deletion-dependent serum alanine aminotransferase elevation, and genotype independently augmented the hepatic post-IR GSK-3β phosphorylation response. These findings support a role for KCNE4 as a systemic modulator of IR injury response and uncover hormonally influenced, sex-specific, KCNE4-dependent and -independent RISK/SAFE pathway induction.

BackgroundAberrations in cyclooxygenase and lipoxygenase (LOX) pathways in non-neoplastic, normal appearing mucosa from patients with colorectal neoplasia (CRN), could hypothetically qualify as predisposing CRN-markers. To test this hypothesis, biopsies were obtained during colonoscopy from macroscopically normal colonic mucosa from patients with and without CRN. Prostaglandin E2 (PGE2) receptors, EP1-4, were examined in Ussing-chambers by exposing biopsies to selective EP receptor agonists, antagonists and PGE2. Furthermore, mRNA expression of EP receptors, prostanoid synthases and LOX enzymes were evaluated using qPCR technology.Results Data suggest that PGE2 binds to high and low affinity EP receptors. In particular, PGE2 demonstrated EP4 receptor potency in the low nanomolar range. Similar results were detected using EP2 and EP4 agonists. In CRN patients, mRNA-levels were higher for EP1 and EP2 receptors and for enzymes prostaglandin-I synthase, 5-LOX, 12-LOX and 15-LOX. ConclusionIn conclusion, normal appearing colonic mucosa from CRN patients demonstrates deviating expression in eicosanoid pathways, indicating a likely predisposition for early CRN development. Moreover, PGE2 potency activates high affinity EP4 receptor subtypes, supporting relevance of testing EP4 antagonists in colorectal cancer management.

In arterial smooth muscle cells, changes in availability of integral membrane proteins influence the regulation of blood flow and blood pressure, which is critical for human health. However, the mechanisms that coordinate the trafficking and membrane expression of specific receptors and ion channels in vascular smooth muscle are poorly understood. In the vasculature, very little is known about microtubules, which form a road network upon which proteins can be transported to and from the cell membrane. This review article summarizes the impact of the microtubule network on arterial contractility, highlighting the importance of the network, with an emphasis on our recent findings regarding the trafficking of the voltage‐dependent Kv7 channels.

About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hKIon channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model.Fourteen resin acid derivatives were tested on hKThe described resin acid derivatives hold promise for new antiseizure medications, with reduced risk for adverse effects compared with retigabine.

van der Horst et al. show that dynein, a microtubule motor protein, carries the voltage-gated potassium channel Kv7.4 away from the cell membrane in vascular smooth muscle cells, thereby reducing its functional impact. These data have implications for our understanding of arterial contractility., 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.

Objective About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hK(V)) channel hK(V)7.2/7.3 is a validated antiseizure target for compounds that activate this channel. In a previous study we have shown that resin acid derivatives can activate the hK(V)7.2/7.3 channel. In this study we investigated if these channel activators have the potential to be developed into a new type of antiseizure drug. Thus we examined their structure-activity relationships and the site of action on the hK(V)7.2/7.3 channel, if they have unwanted cardiac and cardiovascular effects, and their potential antiseizure effect.Methods Ion channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model.Results Fourteen resin acid derivatives were tested on hK(V)7.2/7.3. The most efficient channel activators were halogenated and had a permanently negatively charged sulfonyl group. The compounds did not bind to the sites of other hK(V)7.2/7.3 channel activators, retigabine, or ICA-069673. Instead, they interacted with the most extracellular gating charge of the S4 voltage-sensing helix, and the effects are consistent with an electrostatic mechanism. The compounds altered the voltage dependence of hK(V)7.4, but in contrast to retigabine, there were no effects on the maximum conductance. Consistent with these data, the compounds had less smooth muscle-relaxing effect than retigabine. The compounds had almost no effect on the voltage dependence of hK(V)11.1, hNa(V)1.5, or hCa(V)1.2, or on the amplitude of hK(V)11.1. Finally, several resin acid derivatives had clear antiseizure effects in a zebrafish-larvae model.Significance The described resin acid derivatives hold promise for new antiseizure medications, with reduced risk for adverse effects compared with retigabine.

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.

KCNE5 is an X-linked gene encoding KCNE5, an ancillary subunit to voltage-gated potassium (K-V) channels. Human KCNE5 mutations are associated with atrial fibrillation (AF)- and Brugada syndrome (BrS)-induced cardiac arrhythmias that can arise from increased potassium current in cardiomyocytes. Seeking to establish underlying molecular mechanisms, we created and studied Kcne5 knockout (Kcne5(-/0)) mice. Intracardiac ECG revealed that Kcne5 deletion caused ventricular premature beats, increased susceptibility to induction of polymorphic ventricular tachycardia (60 vs. 24% in Kcne5(+/0) mice), and 10% shorter ventricular refractory period. Kcne5 deletion increased mean ventricular myocyte K-V current density in the apex and also in the subpopulation of septal myocytes that lack fast transient outward current (I-to,I-f). The current increases arose from an apex-specific increase in slow transient outward current-1 (I-Kslow,I-1) (conducted by K(V)1.5) and I-to,I-f (conducted by K(V)4) and an increase in I-Kslow,I-2 (conducted by K(V)2.1) in both apex and septum. Kcne5 protein localized to the intercalated discs in ventricular myocytes, where K(V)2.1 was also detected in both Kcne5(-/0) and Kcne5(+/0) mice. In HL-1 cardiac cells and human embryonic kidney cells, KCNE5 and K(V)2.1 colocalized at the cell surface, but predominantly in intracellular vesicles, suggesting that Kcne5 deletion increases I-K,I-slow2 by reducing K(V)2.1 intracellular sequestration. The human AF-associated mutation KCNE5-L65F negative shifted the voltage dependence of K(V)2.1-KCNE5 channels, increasing their maximum current density >2-fold, whereas BrS-associated KCNE5 mutations produced more subtle negative shifts in K(V)2.1 voltage dependence. The findings represent the first reported native role for Kcne5 and the first demonstrated Kcne regulation of K(V)2.1 in mouse heart. Increased K-V current is a manifestation of KCNE5 disruption that is most likely common to both mouse and human hearts, providing a plausible mechanistic basis for human KCNE5-linked AF and BrS.David, J.-P., Lisewski, U., Crump, S. M., Jepps, T. A., Bocksteins, E., Wilck, N., Lossie, J., Roepke, T. K., Schmitt, N., Abbott, G. W. Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular K-V currents and predisposes to ventricular arrhythmia.

Resistance arteries are small blood vessels that create resistance to blood flow. In hypertension, resistance arteries undergo remodeling, affecting their ability to contract and relax appropriately. To date, no study has mapped the hypertension-related proteomic changes in resistance arteries. Using a novel data-independent acquisition-mass spectrometry (DIA-MS) approach, we determined the proteomic changes in small mesenteric and renal arteries in pre- and early-onset hypertension from the spontaneously hypertensive rat (SHR) model, which represents human primary hypertension. Compared with normotensive controls, mesenteric arteries from 12-week-old SHRs had 286 proteins that were significantly up- or downregulated, whereas 52 proteins were identified as up- or downregulated in mesenteric arteries from 6-week-old SHRs. Of these proteins, 18 were also similarly regulated in SHR renal arteries. Our pathway analyses reveal several novel pathways in the pathogenesis of hypertension. Finally, using a matrisome database, we identified 38 altered extracellular-matrix-associated proteins, many of which have never previously been associated with hypertension. Taken together, this study reveals novel proteins and mechanisms that are associated with early-onset hypertension, thereby providing novel insights into disease progression.

Background and purpose Kv 7.4 and Kv 7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (KV ) channels, with little inhibitory effect on Kv 7 family members at mmol concentrations. However, the effect of 4-AP on Kv 7 channels has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on Kv 7.4 and Kv 7.5 channels and characterize the effect of 4-AP on rat resistance arteries. Experimental approach Voltage clamp experiments were performed on Xenopus laevis oocytes injected with cRNA encoding KCNQ4 or KCNQ5, HEK cells expressing Kv 7.4 channels and on rat, freshly isolated mesenteric artery smooth muscle cells. The effect of 4-AP on tension, membrane potential, intracellular calcium and pH was assessed in rat mesenteric artery segments. Key results 4-AP increased the Kv 7.4-mediated current in oocytes and HEK cells but did not affect Kv 7.5 current. 4-AP also enhanced native mesenteric artery myocyte K+ current at sub-mmol concentrations. When applied to NA-preconstricted mesenteric artery segments, 4-AP hyperpolarized the membrane, decreased [Ca2+ ]i and caused concentration-dependent relaxations that were independent of 4-AP-mediated changes in intracellular pH. Application of the Kv 7 channel blocker XE991 and BKCa channel blocker iberiotoxin attenuated 4-AP-mediated relaxation. 4-AP also inhibited the NA-mediated signal transduction to elicit a relaxation. Conclusions and implications These data show that 4-AP is able to relax NA-preconstricted rat mesenteric arteries by enhancing the activity of Kv 7.4 and BKCa channels and attenuating NA-mediated signalling.

Aims Syncope is a common condition associated with frequent hospitalization or visits to the emergency department. Family aggregation and twin studies have shown that syncope has a heritable component. We investigated whether common genetic variants predispose to syncope and collapse. Methods and results We used genome-wide association data on syncope on 408 961 individuals with European ancestry from the UK Biobank study. In a replication study, we used the Integrative Psychiatric Research Consortium (iPSYCH) cohort (n = 86 189), to investigate the risk of incident syncope stratified by genotype carrier status. We report on a genome-wide significant locus located on chromosome 2q32.1 [odds ratio = 1.13, 95% confidence interval (CI) 1.10–1.17, P = 5.8 × 10−15], with lead single nucleotide polymorphism rs12465214 in proximity to the gene zinc finger protein 804a (ZNF804A). This association was also shown in the iPSYCH cohort, where homozygous carriers of the C allele conferred an increased hazard ratio (1.30, 95% CI 1.15–1.46, P = 1.68 × 10−5) of incident syncope. Quantitative polymerase chain reaction analysis showed ZNF804A to be expressed most abundantly in brain tissue. Conclusion We identified a genome-wide significant locus (rs12465214) associated with syncope and collapse. The association was replicated in an independent cohort. This is the first genome-wide association study to associate a locus with syncope and collapse.

Botanical folk medicines have been used throughout human history to treat common disorders such as hypertension, often with unknown underlying mechanisms. Here, we discovered that hypotensive folk medicines from a genetically diverse range of plant species each selectively activated the vascular-expressed KCNQ5 potassium channel, a feature lacking in the modern synthetic pharmacopeia, whereas nonhypotensive plant extracts did not. Analyzing constituents of the hypotensive Sophora flavescens root, we found that the quinolizidine alkaloid aloperine is a KCNQ-dependent vasorelaxant that potently and isoform-selectively activates KCNQ5 by binding near the foot of the channel voltage sensor. Our findings reveal that KCNQ5-selective activation is a defining molecular mechanistic signature of genetically diverse traditional botanical hypotensives, transcending plant genus and human cultural boundaries. Discovery of botanical KCNQ5-selective potassium channel openers may enable future targeted therapies for diseases including hypertension and KCNQ5 loss-of-function encephalopathy.

Background Syncope is a common condition in the general population causing frequent hospitalisation and visits to the emergency department. Family aggregation and twin studies have previously indicated that syncope and collapse has a heritable component. Purpose We investigated whether common genetic variants predispose to syncope and collapse. Methods We used genome-wide association data on syncope and collapse for 408,961 individuals with European ancestry from the UK Biobank study. In a replication study, the Integrative Psychiatric Research Consortium (iPSYCH) cohort (n=86,189) was used to investigate the risk of incident syncope stratified by genotype carrier status. Results We report on a genome-wide significant locus on chromosome 2q32.1 with the lead SNP rs12465214 (odds ratio [OR] = 1.13, 95% confidence interval [CI] = 1.10–1.17, P=5.8x10–15; Figure 1a). This association was replicated in the iPSYCH cohort, where homozygous carriers of the C allele conferred an increased hazard ratio (HR=1.30, CI: 1.15–1.46, P=1.68x10–5; Figure 1b). LD score regression demonstrated a significant genetic correlation (rg) with coronary artery disease (rg=0.41, P=6.99x10–15) and related phenotypes such as angina and hypertension (Figure 1c). Analyses of eQTL (P=4x10–8) and epigenetic chromatin states revealed that variation in this locus likely affects expression of the gene ZNF804A, which resides in its proximity (Figure 1d). A qPCR analysis showed that ZNF804A was mostly expressed in the brain. A lower level of ZNF804A expression was also detected in the cerebral arteries. ZNF804A was not expressed in heart tissue. Figure 1 Conclusion rs12465214 is associated with syncope and collapse. Variation in this locus likely modulates the expression of the nearby gene ZNF804A through eQTLs and chromatin interactions. ZNF804A is mainly expressed in the brain and cerebral arteries. However, the precise function of ZNF804A is unknown. Furthermore, syncope and collapse is a polygenetic trait and share a significant genetic overlap with coronary artery disease, angina and hypertension. Acknowledgement/Funding This work was supported by grants from The John and Birthe Meyer Foundation, The Research Foundation of the Heart Centre, Rigshospitalet, The Research

Chronic low-grade inflammation and oxidative damage are strongly associated with pathologies including cardiovascular disease. As a consequence, there is considerable interest in agents that mitigate damage. Selenium compounds can act as potent protective agents against oxidation due to the high reactivity and nucleophilicity of the selenium atom. 1,4-Anhydro-4-seleno- d -talitol (SeTal, a novel water-soluble selenium-based sugar) is a potent oxidant scavenger in vitro and in human plasma. Here we show that SeTal is highly stable in solutions that mimic biological fluids and the gastrointestinal tract, and is not rapidly degraded or metabolized unlike some other selenium-containing compounds. SeTal remains intact during extended storage, and it rapidly penetrates into, and effluxes from, primary human coronary artery endothelial and smooth muscle cells, but does not induce loss of metabolic activity, or modulate cell survival and growth rates at concentrations ≤2 mM. Steady-state intracellular concentrations can reach 2–10 μM. SeTal affords protection against H2O2- and HOCl-mediated oxidative damage, with this being independent of the concentration or activities of the selenium-dependent protective enzymes TrxR and GPx. Protection was observed with both concurrent drug and oxidant administration and also (to a lesser extent) with cellular pre-loading. SeTal also affords protection to isolated arterial segments, with the compound decreasing HOCl (50 μΜ) mediated effects on aortic ring relaxation, consistent with the preservation of NO bioavailability. The stability, bioavailability and protective actions of this compound, suggest that it is worthy of further investigation as a protective agent, particularly in the area of cardiovascular disease.

Background and Purpose KCNQ-encoded voltage-dependent potassium channels (Kv7) are involved in the regulation of vascular tone. In this study we evaluated the influence of Kv7 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 Kv7.4 and Kv7.5 were found in penile arteries, penile veins and corpus cavernosum. The Kv7.2–7.5 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 Kv7 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 Kv7 channels play a role in erectile function and contribute to the pathophysiology of erectile dysfunction, an early indicator of cardiovascular disease.

In tauopathies, such as Alzheimer's disease with or without concomitant amyloid beta plaques, cerebral arteries display pathological remodeling, leading to reduced brain tissue oxygenation and cognitive impairment. The precise mechanisms that underlie this vascular dysfunction remain unclear. Kv7 voltage-dependent K+ channels contribute to the development of myogenic tone in rat cerebral arteries. Thus, we hypothesized that Kv7 channel function would be impaired in the cerebral arteries of a tauopathy mouse model (rTg4510), which might underlie cerebral hypoperfusion associated with the development of neurofibrillary tangles in tauopathies. To test our hypothesis we performed wire myography and quantitative PCR on cerebral arteries, mesenteric arteries and the inferior frontotemporal region of the brain surrounding the middle cerebral artery from tau transgenic mice (rTg4510) and aged-matched controls. We also performed whole-cell patch clamp experiments on HEK293 cells stably expressing Kv7.4. Here, we show that Kv7 channels are functionally impaired in the cerebral arteries of rTg4510 mice, but not in mesenteric arteries from the same mice. The quantitative PCR analysis of the cerebral arteries found no change in the expression of the genes encoding the Kv7 channel alpha-subunits, however, we found reduced expression of the ancillary subunit, KCNE5 (also termed KCNEIL), in the cerebral arteries of rTg4510 mice. In the brain, rTg4510 mice showed reduced expression of Kv7.3, Kv7.5, and Kv2.1. Co-expression of KCNE5 with Kv7.4 in HEK293 cells produced larger currents at voltages >0 mV and increased the deactivation time for the Kv7.4 channel. Thus, our results demonstrate that Kv7 channel function is attenuated in the cerebral arteries of Tg4510 mice, which may result from decreased KCNE5 expression. Reduced Kv7 channel function might contribute to cerebral hypoperfusion in tauopathies, such as Alzheimer's disease.

KCNE5 is an X-linked gene encoding KCNE5, an ancillary subunit to voltage-gated potassium (K(V)) channels. Human KCNE5 mutations are associated with atrial fibrillation (AF)– and Brugada syndrome (BrS)–induced cardiac arrhythmias that can arise from increased potassium current in cardiomyocytes. Seeking to establish underlying molecular mechanisms, we created and studied Kcne5 knockout (Kcne5(−/0)) mice. Intracardiac ECG revealed that Kcne5 deletion caused ventricular premature beats, increased susceptibility to induction of polymorphic ventricular tachycardia (60 vs. 24% in Kcne5(+/0) mice), and 10% shorter ventricular refractory period. Kcne5 deletion increased mean ventricular myocyte K(V) current density in the apex and also in the subpopulation of septal myocytes that lack fast transient outward current (I(to,f)). The current increases arose from an apex-specific increase in slow transient outward current-1 (I(Kslow,1)) (conducted by K(V)1.5) and I(to,f) (conducted by K(V)4) and an increase in I(Kslow,2) (conducted by K(V)2.1) in both apex and septum. Kcne5 protein localized to the intercalated discs in ventricular myocytes, where K(V)2.1 was also detected in both Kcne5(−/0) and Kcne5(+/0) mice. In HL-1 cardiac cells and human embryonic kidney cells, KCNE5 and K(V)2.1 colocalized at the cell surface, but predominantly in intracellular vesicles, suggesting that Kcne5 deletion increases I(K,slow2) by reducing K(V)2.1 intracellular sequestration. The human AF-associated mutation KCNE5-L65F negative shifted the voltage dependence of K(V)2.1-KCNE5 channels, increasing their maximum current density >2-fold, whereas BrS-associated KCNE5 mutations produced more subtle negative shifts in K(V)2.1 voltage dependence. The findings represent the first reported native role for Kcne5 and the first demonstrated Kcne regulation of K(V)2.1 in mouse heart. Increased K(V) current is a manifestation of KCNE5 disruption that is most likely common to both mouse and human hearts, providing a plausible mechanistic basis for human KCNE5-linked AF and BrS.—David, J.-P., Lisewski, U., Crump, S. M., Jepps, T. A., Bocksteins, E., Wilck, N., Lossie, J., Roepke, T. K., Schmitt, N., Abbott, G. W. Deletion in mice of X-linked, Brugada syndrome– and atrial fibrillation–associated Kcne5 augments ventricular K(V) currents and predisposes to ventricular arrhythmia.

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. Ca2+‐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 Ca2+‐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, T16Ainh‐A01, and Ani9 attenuated 5‐HT/U46619‐induced contractions. MONNA and T16Ainh‐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.

Voltage gated Kv7.4 channels have been implicated in vascular smooth muscle cells (VSMCs) activity as they modulate basal arterial contractility, mediate responses to endogenous vasorelaxants, and are down-regulated 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 PCR revealed that in whole rat mesenteric artery Ang II incubation for 1-7h 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 VSMCs. 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, enhanced the interaction of K(v)7.4 with the E3 ubiquitin ligase CHIP, and increased K(v)7.4 ubiquitination. Similar alterations were found in mesenteric VSMCs isolated from Ang II-infused mice. The effect of Ang II was emulated by 17-AAG 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.

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.

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.

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.

Human proximal and distal ureter tissues were studied to clarify whether the presence of mucosa affects contractile responses. In histological studies, human ureter was compared with urinary bladder (detrusor). Contractions in response to high KCl solution, phenylephrine, and carbachol were measured in intact and mucosa-denuded strips of human ureter. Tissue sections of human bladder and ureter were used for histological staining. Thirty-four percent of the ureter strips contracted spontaneously with highly variable patterns, and this was affected neither by mucosa nor by proximal or distal tissue origin. Upon stimulation with 40 mM KCl, ureter strips exhibited strong phasic and weak tonic contractions. In intact strips, normalized tonic force was lower than in denuded strips, but no consistent effect of mucosa was observed with phasic contractions. Absolute force values of phasic contractions were weaker in proximal than distal ureter strips, but similar when normalized to tissue wet weight. Stimulation with 80 mM KCl enhanced tonic contraction fourfold; phasic contractions occurred rarely. Phenylephrine produced no statistically significant stronger tonic contraction in distal compared with proximal ureter strips; nevertheless, in some strips, pre-existing spontaneous contractions increased. Carbachol did not influence ureter contractions. In the bladder, a suburothelial cell layer stained positive with α-smooth muscle actin (α-SMA)-specific antibodies could be further differentiated with vimentin- and desmin-specific antibodies. α-SMA positive cells were absent in suburothelial ureter tissue. Like in detrusor, the mucosa inhibits KCl-stimulated tonic ureter contractions. The mucosa of detrusor and ureter tissue exhibits distinct staining patterns for α-SMA, vimentin, and desmin. This suggests a different distribution of smooth muscle cells, fibroblasts, and myofibroblasts, which could be a target for pharmacological therapy of pathologic contractile processes.

Microtubules can regulate GPCR (G protein–coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance of this pathway in vascular smooth muscle cells, we investigated the role of microtubule stability on β-adrenoceptor signaling in rat renal and mesenteric arteries. In isometric tension experiments, incubation with the microtubule inhibitors colchicine and nocodazole enhanced isoprenaline-mediated relaxations of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated the effect of colchicine on isoprenaline relaxations, whereas iberiotoxin—a BKCa channel blocker—had no effect. In addition, colchicine improved the relaxations to the Kv7.2 to 7.5 activator, S-1, in both renal and mesenteric artery segments compared with dimethyl sulfoxide incubation. We determined that increased mesenteric artery myocytes treated with colchicine showed increased Kv7.4 membrane expression, but Western blot analysis showed no change in total Kv7.4 protein. This study is the first to show microtubule disruption improves the β-adrenoceptor–mediated relaxations of mesenteric and renal arteries and determine this enhancement to be because of increased membrane expression of the Kv7 voltage-gated potassium channels.

Which ion channel is the most important for regulating vascular tone? Which one is responsible for controlling the resting membrane potential or repolarization? Which channels are recruited by different intracellular signalling pathways or change in certain vascular diseases? Many different ion channels have been identified in the vasculature over the years and claimed as future therapeutic targets. Unfortunately, several of these ion channels are not just found in the vasculature, with many of them also found to have prominent functional roles in different organs of the body, which then leads to off-target effects. As cardiovascular diseases are expected to increase worldwide to epidemic proportions, ion channel research and the hunt for the next major therapeutic target to treat different vascular diseases has never been more important. However, I believe that the question we should now be asking is: which ancillary subunits are involved in regulating specific ion channels in the vasculature and do they have the potential to be new therapeutic targets?

Background and Purpose The KCNQ-encoded voltage-gated potassium channel family (Kv7.1-Kv7.5) are established regulators of smooth muscle contractility, where Kv7.4 and Kv7.5 predominate. Various Kv7.2–7.5 channel enhancers have been developed that have been shown to cause a vasorelaxation in both rodent and human blood vessels. Recently, two novel Kv7 channel enhancers have been identified, ML213 and NS15370, that show increased potency, particularly on Kv7.4 channels. The aim of this study was to characterize the effects of these novel enhancers in different rat blood vessels and compare them with Kv7 enhancers (S-1, BMS204352, retigabine) described previously. We also sought to determine the binding sites of the new Kv7 enhancers. Key Results Both ML213 and NS15370 relaxed segments of rat thoracic aorta, renal artery and mesenteric artery in a concentration-dependent manner. In the mesenteric artery ML213 and NS15370 displayed EC50s that were far lower than other Kv7 enhancers tested. Current-clamp experiments revealed that both novel enhancers, at low concentrations, caused significant hyperpolarization in mesenteric artery smooth muscle cells. In addition, we determined that the stimulatory effect of these enhancers relied on a tryptophan residue located in the S5 domain, which is the same binding site for the other Kv7 enhancers tested in this study. Conclusions and Implications This study has identified and characterized ML213 and NS15370 as potent vasorelaxants in different blood vessels, thereby highlighting these new compounds as potential therapeutics for various smooth muscle disorders.

Voltage-gated potassium (Kv) channels formed by Kv7 (KCNQ) α-subunits are recognized as crucial for vascular smooth muscle function, in addition to their established roles in the heart (Kv7.1) and the brain (Kv7.2-5). In vivo, Kv7 α-subunits are often regulated by KCNE subfamily ancillary (β) subunits. We investigated the effects of targeted germline Kcne4 deletion on mesenteric artery reactivity in adult male and female mice. Kcne4 deletion increased mesenteric artery contractility in response to α-adrenoceptor agonist methoxamine, and decreased responses to Kv7.2-7.5 channel activator ML213, in male but not female mice. In contrast, Kcne4 deletion markedly decreased vasorelaxation in response to isoprenaline in both male and female mice. Kcne4 expression was 2-fold lower in the female versus the male mouse mesenteric artery, and Kcne4 deletion elicited only moderate changes of other Kcne transcripts, with no striking sex-specific differences. However, Kv7.4 protein expression in females was twice that in males, and was reduced in both sexes by Kcne4 deletion. Our findings confirm a crucial role for KCNE4 in regulation of Kv7 channel activity to modulate vascular tone, and provide the first known molecular mechanism for sex-specificity of this modulation that has important implications for vascular reactivity and may underlie sex-specific susceptibility to cardiovascular diseases.