Proximity Labelling to Quantify Kv7.4 and Dynein Protein Interaction in Freshly Isolated Rat Vascular Smooth Muscle Cells.

Understanding protein-protein interactions is crucial for unravelling subcellular protein distribution, contributing to our understanding of cellular organisation. Moreover, interaction studies can reveal insights into the mechanisms that cover protein trafficking within cells. Although various techniques such as Förster resonance energy transfer (FRET), co-immunoprecipitation, and fluorescence microscopy are commonly employed to detect protein interactions, their limitations have led to more advanced techniques such as the in situ proximity ligation assay (PLA) for spatial co-localisation analysis. The PLA technique, specifically employed in fixed cells and tissues, utilises species-specific secondary PLA probes linked to DNA oligonucleotides. When proteins are within 40 nm of each other, the DNA oligonucleotides on the probes interact, facilitating circular DNA formation through ligation. Rolling-circle amplification then produces DNA circles linked to the PLA probe. Fluorescently labelled oligonucleotides hybridise to the circles, generating detectable signals for precise co-localisation analysis. We employed PLA to examine the co-localisation of dynein with the Kv7.4 channel protein in isolated vascular smooth muscle cells from rat mesenteric arteries. This method enabled us to investigate whether Kv7.4 channels interact with dynein, thereby providing evidence of their retrograde transport by the microtubule network. Our findings illustrate that PLA is a valuable tool for studying potential novel protein interactions with dynein, and the quantifiable approach offers insights into whether these interactions are changed in disease.

Suppression of smooth muscle cell inflammation by myocardin-related transcription factors involves inactivation of TANK-binding kinase 1.

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) suppress production of pro-inflammatory cytokines and chemokines in human smooth muscle cells (SMCs) through sequestration of RelA in the NF-κB complex, but additional mechanisms are likely involved. The cGAS-STING pathway is activated by double-stranded DNA in the cytosolic compartment and acts through TANK-binding kinase 1 (TBK1) to spark inflammation. The present study tested if MRTFs suppress inflammation also by targeting cGAS-STING signaling. Interrogation of a transcriptomic dataset where myocardin was overexpressed using a panel of 56 cGAS-STING cytokines showed the panel to be repressed. Moreover, MYOCD, MRTFA, and SRF associated negatively with the panel in human arteries. RT-qPCR in human bronchial SMCs showed that all MRTFs reduced pro-inflammatory cytokines on the panel. MRTFs diminished phosphorylation of TBK1, while STING phosphorylation was marginally affected. The TBK1 inhibitor amlexanox, but not the STING inhibitor H-151, reduced the anti-inflammatory effect of MRTF-A. Co-immunoprecipitation and proximity ligation assays supported binding between MRTF-A and TBK1 in SMCs. MRTFs thus appear to suppress cellular inflammation in part by acting on the kinase TBK1. This may defend SMCs against pro-inflammatory insults in disease.

Mesenteric artery smooth muscle cells from hypertensive rats have increased microtubule acetylation.

The dynamic nature of the microtubule network is dependent in part by post-translational modifications (PTMs) - particularly through acetylation, which stabilizes the microtubule network. Whether PTMs of the microtubule network in vascular smooth muscle cells (VSMCs) contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR). Experiments were performed on male normotensive rats and SHR mesenteric arteries. Western blotting and mass spectrometry determined changes in tubulin acetylation. Wire myography was used to investigate the effect of tubacin on isoprenaline-mediated vasorelaxations. Isolated cells from normotensive rats were used for scanning ion conductance microscopy (SICM). Mass spectrometry and Western blotting showed that tubulin acetylation is increased in the mesenteric arteries of the SHR compared with normotensive rats. Tubacin enhanced the ß-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young's modulus of VSMCs, but found no difference in control, tubacin-treated, or taxol-treated cells. Acetylation of tubulin at Lys40 is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

Crucial role for Sodium Hydrogen Exchangers in SGLT2 inhibitor-induced arterial relaxations.

Introduction: Sodium dependent glucose transporter 2 (SGLT2 or SLC5A2) inhibitors effectively lower blood glucose and are also approved treatments for heart failure independent of raised glucose. One component of the cardioprotective effect is reduced cardiac afterload but the mechanisms underlying peripheral relaxation are ill defined and variable. We speculated that SGLT2 inhibitors promoted arterial relaxation via the release of the potent vasodilator calcitonin gene-related peptide (CGRP) from sensory nerves independent of glucose transport. Experimental approach: The functional effects of SGLT2 inhibitors (dapagliflozin, empagliflozin, ertugliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted mesenteric and renal arteries from male Wistar rats using Wire-Myography. SGLT2, NHE1, CGRP and TRPV1 expression in both arteries 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. Results: All SGLT2 inhibitors produced a concentration dependent relaxation (1µM-100µM) of mesenteric arteries that was considerably greater than in renal arteries. Cariporide relaxed mesenteric arteries but not renal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that was absent in renal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin produced significantly greater relaxations in mesenteric arteries compared to renal arteries. Relaxations to dapagliflozin, empagliflozin and cariporide were attenuated by incubation with the CGRP receptor antagonist BIBN-4096, the Kv7 blocker linopirdine and the TRPV1 antagonist AMG-517 as well as by depletion of neuronal CGRP. Neither dapagliflozin nor empagliflozin directly activated heterologously expressed TRPV1 channels or Kv7 channels. Strikingly, only NHE1 colocalised with TRPV1 in sensory nerves, and cariporide pre-application prevented the relaxant response to SGLT2 inhibitors. Conclusions: SGLT2 inhibitors relax mesenteric arteries by a novel mechanism involving the release of CGRP from sensory nerves following inhibition of the Na + /H + exchanger.

Acute, pro-contractile effects of prorenin on rat mesenteric arteries.

Prorenin and the prorenin receptor ((P)RR) are important, yet controversial, members of the renin-angiotensin-aldosterone system. The ((P)RR) is expressed throughout the body, including the vasculature, however, the direct effect of prorenin on arterial contractility is yet to be determined. Within rat mesenteric arteries, immunostaining and proximity ligation assays were used to determine the interacting partners of (P)RR in freshly isolated vascular smooth muscle cells (VSMCs). Wire myography examined the functional effect of prorenin. Simultaneous changes in [Ca2+ ]i and force were recorded in arteries loaded with Fura-2AM. Spontaneously transient outward currents were recorded via perforated whole-cell patch-clamp configuration in freshly isolated VSMCs. We found that the (P)RR is located within a distance of less than 40 nm from the V-ATPase, caveolin-1, ryanodine receptors, and large conductance Ca2+ -activated K+ channels (BKCa ) in VSMCs. [Ca2+ ]i imaging and isometric tension recordings indicate that 1 nM prorenin enhanced α1-adrenoreceptor-mediated contraction, associated with an increased number of Ca2+ waves, independent of voltage-gated Ca2+ channels activation. Incubation of VSMCs with 1 nM prorenin decreased the amplitude and frequency of spontaneously transient outward currents and attenuated BKCa -mediated relaxation. Inhibition of the V-ATPase with 100 nM bafilomycin prevented prorenin-mediated inhibition of BKCa -derived relaxation. Renin (1 nM) had no effect on BKCa -mediated relaxation. In conclusion, prorenin enhances arterial contractility by inhibition of BKCa and increasing intracellular Ca2+ release. It is likely that this effect is mediated through a local shift in pH upon activation of the (P)RR and stimulation of the V-ATPase.

Proteomic mapping reveals dysregulated angiogenesis in the cerebral arteries of rats with early-onset hypertension.

Hypertension is associated with the presence of vascular abnormalities, including 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, here 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 in humans. Our analysis identified 125 proteins with expression levels that were significantly upregulated or downregulated in 12-week-old spontaneously hypertensive rats compared to normotensive Wistar Kyoto rats. Using an angiogenesis enrichment analysis, we further identified a critical imbalance in angiogenic proteins that promoted an anti-angiogenic profile in cerebral arteries at 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 insights into the early pathology of cerebrovascular disease.

Vascular smooth muscle-specific YAP/TAZ deletion triggers aneurysm development in mouse aorta.

Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8-Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.

Medicinal plant rosemary relaxes blood vessels by activating vascular smooth muscle KCNQ channels.

The evergreen plant rosemary (Salvia rosmarinus) has been employed medicinally for centuries as a memory aid, analgesic, spasmolytic, vasorelaxant and antihypertensive, with recent preclinical and clinical evidence rationalizing some applications. Voltage-gated potassium (Kv) channels in the KCNQ (Kv7) subfamily are highly influential in the nervous system, muscle and epithelia. KCNQ4 and KCNQ5 regulate vascular smooth muscle excitability and contractility and are implicated as antihypertensive drug targets. Here, we found that rosemary extract potentiates homomeric and heteromeric KCNQ4 and KCNQ5 activity, resulting in membrane hyperpolarization. Two rosemary diterpenes, carnosol and carnosic acid, underlie the effects and, like rosemary, are efficacious KCNQ-dependent vasorelaxants, quantified by myography in rat mesenteric arteries. Sex- and estrous cycle stage-dependence of the vasorelaxation matches sex- and estrous cycle stage-dependent KCNQ expression. The results uncover a molecular mechanism underlying rosemary vasorelaxant effects and identify new chemical spaces for KCNQ-dependent vasorelaxants.

Cycling matters: Sex hormone regulation of vascular potassium channels.

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.

Colchicine enhances ß adrenoceptor-mediated vasodilation in men with essential hypertension.

AIMS: The aim of this study is to examine whether colchicine improves ß 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 ß 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 ß adrenoceptor response, and there was no effect on arterial pressure, arterial compliance or the level of measured inflammatory markers. CONCLUSION: Colchicine acutely enhances ß 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 ß adrenoceptor-mediated vasodilation in humans with essential hypertension. CONDENSED ABSTRACT: Preclinical studies in isolated rodent arteries have shown that colchicine can enhance ß 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 ß 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.

The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells.

Background: Several local Ca2+ events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca2+ transients near the plasma membrane, and suggested the importance of this signaling for regulation of intercellular coupling and smooth muscle cell contraction. The mechanism behind these Na,K-ATPase-dependent "Ca2+ flashes" remains to be elucidated. In addition to its conventional ion transport function, the Na,K-ATPase is proposed to contribute to intracellular pathways, including Src kinase activation. The microtubule network is important for intracellular signaling, but its role in the Na,K-ATPase-Src kinase interaction is not known. We hypothesized the microtubule network was responsible for maintaining the Na,K-ATPase-Src kinase interaction, which enables Ca2+ flashes. Methods: We characterized Ca2+ flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca2+-sensitive fluorescent dyes, Calcium Green-1/AM and Fura Red/AM, for ratiometric measurements of intracellular Ca2+. The Na,K-ATPase α2 isoform was knocked down with siRNA and the microtubule network was disrupted with nocodazole. An involvement of the Src signaling was tested pharmacologically and with Western blot. Protein interactions were validated with proximity ligation assays. Results: The Ca2+ flashes were induced by micromolar concentrations of ouabain. Knockdown of the α2 isoform Na,K-ATPase abolished Ca2+ flashes, as did inhibition of tyrosine phosphorylation with genistein and PP2, and the inhibitor of the Na,K-ATPase-dependent Src activation, pNaKtide. Ouabain-induced Ca2+ flashes were associated with Src kinase activation by phosphorylation. The α2 isoform Na,K-ATPase and Src kinase colocalized in the cells. Disruption of microtubule with nocodazole inhibited Ca2+ flashes, reduced Na,K-ATPase/Src interaction and Src activation. Conclusion: We demonstrate that the Na,K-ATPase-dependent Ca2+ flashes in smooth muscle cells require an interaction between the α2 isoform Na, K-ATPase and Src kinase, which is maintained by the microtubule network.

Dynein Coordinates ß2-Adrenoceptor-Mediated Relaxation in Normotensive and Hypertensive Rat Mesenteric Arteries.

BACKGROUND: The voltage-gated potassium channel (Kv)7.4 and Kv7.5 channels contribute to the ß-adrenoceptor-mediated vasodilatation. In arteries from hypertensive rodents, the Kv7.4 channel is downregulated and function attenuated, which contributes to the reduced ß-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 ß-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 ß-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 ß2-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 ß2-adrenoceptor following isoprenaline treatment. Immunostaining experiments showed ciliobrevin D prevented isoprenaline-mediated internalizationof the ß2-adrenoceptor. CONCLUSIONS: Overall, these data show that colchicine and ciliobrevin D can induce a ß2-adrenoceptor-mediated vasodilatation in arteries from the spontaneously hypertensive rat as well as reinstating Kv7.4 channel function.

KCNQ5 activation by tannins mediates vasorelaxant effects of barks used in Native American botanical medicine.

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.

Identification of novel proteins and mechanistic pathways associated with early-onset hypertension by deep proteomic mapping of resistance arteries.

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.

Functional sympatholysis in mouse skeletal muscle involves sarcoplasmic reticulum swelling in arterial smooth muscle cells.

The vasoconstrictive effect of sympathetic activity is attenuated in contracting skeletal muscle (functional sympatholysis), allowing increased blood supply to the working muscle but the underlying mechanisms are incompletely understood. The purpose of this study was to examine α-adrenergic receptor responsiveness in isolated artery segments from non-exercised and exercised mice, using wire myography. Isometric tension recordings performed on femoral artery segments from exercised mice showed decreased α-adrenergic receptor responsiveness compared to non-exercised mice (logEC50 -5.2 ± 0.04 M vs. -5.7 ± 0.08 M, respectively). In contrast, mesenteric artery segments from exercised mice displayed similar α-adrenergic receptor responses compared to non-exercised mice. Responses to the vasoconstrictor serotonin (5-HT) and vasodilator isoprenaline, were similar in femoral artery segments from non-exercised and exercised mice. To study sarcoplasmic reticulum (SR) function, we examined arterial contractions induced by caffeine, which depletes SR Ca2+ and thapsigargin, which inhibits SR Ca2+ -ATPase (SERCA) and SR Ca2+ uptake. Arterial contractions to both caffeine and thapsigargin were increased in femoral artery segment from exercised compared to non-exercised mice. Furthermore, 3D electron microscopy imaging of the arterial wall showed SR volume/length ratio increased 157% in smooth muscle cells of the femoral artery from the exercised mice, whereas there was no difference in SR volume/length ratio in mesenteric artery segments. These results show that in arteries surrounding exercising muscle, the α-adrenergic receptor constrictions are blunted, which can be attributed to swollen smooth muscle cell SR's, likely due to increased Ca2+ content that is possibly reducing free intracellular Ca2+ available for contraction. Overall, this study uncovers a previously unknown mechanism underlying functional sympatholysis.

Kv7 channel trafficking by the microtubule network in vascular smooth muscle.

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.

KCNQ5 Potassium Channel Activation Underlies Vasodilation by Tea.

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.

Dynein regulates Kv7.4 channel trafficking from the cell membrane.

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.