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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.

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.

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.

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.

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.

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.

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.

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.