G protein ßγ regulation of KCNQ-encoded voltage-dependent K channels.

The KCNQ family is comprised of five genes and the expression products form voltage-gated potassium channels (Kv7.1-7.5) that have a major impact upon cellular physiology in many cell types. Each functional Kv7 channel forms as a tetramer that often associates with proteins encoded by the KCNE gene family (KCNE1-5) and is critically reliant upon binding of phosphatidylinositol bisphosphate (PIP2) and calmodulin. Other modulators like A-kinase anchoring proteins, ubiquitin ligases and Ca-calmodulin kinase II alter Kv7 channel function and trafficking in an isoform specific manner. It has now been identified that for Kv7.4, G protein ßγ subunits (Gßγ) can be added to the list of key regulators and is paramount for channel activity. This article provides an overview of this nascent field of research, highlighting themes and directions for future study.

Marked oestrous cycle-dependent regulation of rat arterial KV 7.4 channels driven by GPER1.

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

KV7 Channel Expression and Function Within Rat Mesenteric Endothelial Cells.

Background and Purpose: Arterial diameter is dictated by the contractile state of the vascular smooth muscle cells (VSMCs), which is modulated by direct and indirect inputs from endothelial cells (ECs). Modulators of KCNQ-encoded kV7 channels have considerable impact on arterial diameter and these channels are known to be expressed in VSMCs but not yet defined in ECs. However, expression of kV7 channels in ECs would add an extra level of vascular control. This study aims to characterize the expression and function of KV7 channels within rat mesenteric artery ECs. Experimental Approach: In rat mesenteric artery, KCNQ transcript and KV7 channel protein expression were determined via RT-qPCR, immunocytochemistry, immunohistochemistry and immunoelectron microscopy. Wire myography was used to determine vascular reactivity. Key Results: KCNQ transcript was identified in isolated ECs and VSMCs. KV7.1, KV7.4 and KV7.5 protein expression was determined in both isolated EC and VSMC and in whole vessels. Removal of ECs attenuated vasorelaxation to two structurally different KV7.2-5 activators S-1 and ML213. KIR2 blockers ML133, and BaCl2 also attenuated S-1 or ML213-mediated vasorelaxation in an endothelium-dependent process. KV7 inhibition attenuated receptor-dependent nitric oxide (NO)-mediated vasorelaxation to carbachol, but had no impact on relaxation to the NO donor, SNP. Conclusion and Implications: In rat mesenteric artery ECs, KV7.4 and KV7.5 channels are expressed, functionally interact with endothelial KIR2.x channels and contribute to endogenous eNOS-mediated relaxation. This study identifies KV7 channels as novel functional channels within rat mesenteric ECs and suggests that these channels are involved in NO release from the endothelium of these vessels.

SMIT (Sodium-Myo-Inositol Transporter) 1 Regulates Arterial Contractility Through the Modulation of Vascular Kv7 Channels.

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

The Gß1 and Gß3 Subunits Differentially Regulate Rat Vascular Kv7 Channels.

Within the vasculature Kv7 channels are key regulators of basal tone and contribute to a variety of receptor mediated vasorelaxants. The Kv7.4 isoform, abundant within the vasculature, is key to these processes and was recently shown to have an obligatory requirement of G-protein ßγ subunits for its voltage dependent activity. There is an increasing appreciation that with 5 Gß subunits and 12 Gγ subunits described in mammalian cells that different Gß x γ x combinations can confer selectivity in Gßγ effector stimulation. Therefore, we aimed to characterize the Gß subunit(s) which basally regulate Kv7.4 channels and native vascular Kv7 channels. In Chinese Hamster Ovary cells overexpressing Kv7.4 and different Gßx subunits only Gß1, Gß3, and Gß5 enhanced Kv7.4 currents, increasing the activation kinetics and negatively shifting the voltage dependence of activation. In isolated rat renal artery myocytes, proximity ligation assay detected an interaction of Kv7.4 with Gß1 and Gß3 subunits, but not other isoforms. Morpholino directed knockdown of Gß1 in rat renal arteries did not alter Kv7 dependent currents but reduced Kv7.4 protein expression. Knockdown of Gß3 in rat renal arteries resulted in decreased basal K+ currents which were not sensitive to pharmacological inhibition of Kv7 channels. These studies implicate the Gß1 subunit in the synthesis or stability of Kv7.4 proteins, whilst revealing that the Gß3 isoform is responsible for the basal activity of Kv7 channels in native rat renal myocytes. These findings demonstrate that different Gß subunits have important individual roles in ion channel regulation.

Investigating the Role of G Protein ßγ in Kv7-Dependent Relaxations of the Rat Vasculature.

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

Angiotensin II Promotes KV7.4 Channels Degradation Through Reduced Interaction With HSP90 (Heat Shock Protein 90).

Voltage-gated Kv7.4 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 Kv7.4. Western blot and quantitative polymerase chain reaction revealed that in whole rat mesenteric artery, Ang II incubation for 1 to 7 hours decreased Kv7.4 protein expression without reducing transcript levels. Moreover, Ang II decreased XE991 (Kv7)-sensitive currents and attenuated membrane potential hyperpolarization and relaxation induced by the Kv7 activator ML213. Ang II also reduced Kv7.4 staining at the plasma membrane of vascular smooth muscle cells. Proteasome inhibition with MG132 prevented Ang II-induced decrease of Kv7.4 levels and counteracted the functional impairment of ML213-induced relaxation in myography experiments. Proximity ligation assays showed that Ang II impaired the interaction of Kv7.4 with the molecular chaperone HSP90 (heat shock protein 90), enhanced the interaction of Kv7.4 with the E3 ubiquitin ligase CHIP (C terminus of Hsp70-interacting protein), and increased Kv7.4 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 Kv7.4 by altering protein stability through a decrease of its interaction with HSP90. This leads to the recruitment of CHIP and Kv7.4 ubiquitination and degradation via the proteasome.

KCNQ-Encoded Potassium Channels as Therapeutic Targets.

Kv7 channels are voltage-gated potassium channels encoded by KCNQ genes that have a considerable physiological impact in many cell types. This reliance upon Kv7 channels for normal cellular function, as well as the existence of hereditary disorders caused by mutations to KCNQ genes, means that pharmacological targeting of these channels has broad appeal. Consequently, a plethora of chemical entities that modulate Kv7 channel activity have been developed. Moreover, Kv7 channels are influenced by many disparate intracellular mediators and trafficking processes, making upstream targeting an appealing prospect for therapeutic development. This review covers the main characteristics of these multifunctional and versatile channels with the aim of providing insight into the therapeutic value of targeting these channels.

Synergistic interplay of Gßγ and phosphatidylinositol 4,5-bisphosphate dictates Kv7.4 channel activity.

Kv7.4 channels are key determinants of arterial contractility and cochlear mechanosensation that, like all Kv7 channels, have an obligatory requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). ßγ G proteins (Gßγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gßγ increased Kv7.4 open probability through an increased sensitivity to PIP2. In HEK cells stably expressing Kv7.4, PIP2 or Gßγ increased open probability in a concentration dependent manner. Depleting PIP2 prevented any Gßγ-mediated stimulation whilst an array of Gßγ inhibitors prohibited any PIP2-induced current enhancement. A combination of PIP2 and Gßγ at sub-efficacious concentrations increased channel open probability considerably. The stimulatory effects of three Kv7.2-7.5 channel activators were also lost by PIP2 depletion or Gßγ inhibitors. This study alters substantially our understanding of the fundamental processes that dictate Kv7.4 activity, revealing a more complex and subtle paradigm where the reliance on local phosphoinositide is dictated by interaction with Gßγ.

Kv7 Channel Activation Underpins EPAC-Dependent Relaxations of Rat Arteries.

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

MicroRNA-153 targeting of KCNQ4 contributes to vascular dysfunction in hypertension.

AIMS: Kv7.4, a voltage-dependent potassium channel expressed throughout the vasculature, controls arterial contraction and is compromised in hypertension by an unknown mechanism. MicroRNAs (miRs) are post-transcriptional regulators of protein production and are altered in disease states such as hypertension. We investigated whether miRs regulate Kv7.4 expression. METHODS AND RESULTS: In renal and mesenteric arteries (MAs) of the spontaneously hypertensive rat (SHR), Kv7.4 protein decreased compared with the normotensive (NT) rat without a decrease in KCNQ4 mRNA, inferring that Kv7.4 abundance was determined by post-transcriptional regulation. In silico analysis of the 3' UTR of KCNQ4 revealed seed sequences for miR26a, miR133a, miR200b, miR153, miR214, miR218, and let-7d with quantitative polymerase chain reaction showing miR153 increased in those arteries from SHRs that exhibited decreased Kv7.4 levels. Luciferase reporter assays indicated a direct targeting effect of miR153 on the 3' UTR of KCNQ4. Introduction of high levels of miR153 to MAs increased vascular wall thickening and reduced Kv7.4 expression/Kv7 channel function compared with vessels receiving a non-targeting miR, providing a proof of concept of Kv7.4 regulation by miR153. CONCLUSION: This study is the first to define a role for aberrant miR153 contributing to the hypertensive state through targeting of KCNQ4 in an animal model of hypertension, raising the possibility of the use of miR153-related therapies in vascular disease.

G-protein ßγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity.

Kv7.4 channels are a crucial determinant of arterial diameter both at rest and in response to endogenous vasodilators. However, nothing is known about the factors that ensure effective activity of these channels. We report that G-protein ßγ subunits increase the amplitude and activation rate of whole-cell voltage-dependent K(+) currents sensitive to the Kv7 blocker linopirdine in HEK cells heterologously expressing Kv7.4, and in rat renal artery myocytes. In excised patch recordings, Gßγ subunits (2-250 ng /mL) enhanced the open probability of Kv7.4 channels without changing unitary conductance. Kv7 channel activity was also augmented by stimulation of G-protein-coupled receptors. Gallein, an inhibitor of Gßγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gßγ subunit inhibitors (GRK2i and a ß-subunit antibody) abolished Kv7 channel currents in the absence of either Gßγ subunit enrichment or G-protein-coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gßγ subunits colocalized in HEK cells and renal artery smooth muscle cells. Gallein disrupted this colocalization, contracted whole renal arteries to a similar degree as the Kv7 inhibitor linopirdine, and impaired isoproterenol-induced relaxations. Furthermore, mSIRK, which disassociates Gßγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gßγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone.

Contribution of Kv7 channels to natriuretic peptide mediated vasodilation in normal and hypertensive rats.

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

Functional and pharmacological characterization of volume-regulated anion channels in human normal and cystic fibrosis bronchial and nasal epithelial cells.

Volume-regulated anion channels (VRACs) are widely present in various cell types and have important functions ranging from regulatory volume decrease to control of cell proliferation and apoptosis. Here we aimed to compare the biophysical features and pharmacological profiles of VRAC currents in healthy and cystic fibrosis (CF) respiratory epithelial cells in order to characterize these currents both functionally and pharmacologically. Whole-cell electrophysiology was used to characterize the VRAC current in normal (16HBE14o-; HBE) and CF cell lines (CFBE14o-; CFBE), as well as in native human nasal epithelial cells. Application of hypotonic solution produced current responses of similar sizes in both HBE and CFBE cells. Biophysical properties of VRACs, such as instantaneous activation and deactivation upon voltage step, some inactivation at potentials positive to 40 mV and outwardly-rectifying I-V curves, were indistinguishable in both cell types. Extensive pharmacological analysis of the currents revealed a similar pharmacological profile in response to three blockers--NPPB, DCPIB and DIDS. Native primary human nasal epithelial cells from both healthy and CF volunteers also showed typical VRAC responses of comparable sizes. VRACs in these cells were more sensitive to external solution hypotonicity compared to HBE and CFBE cells. In all cell types studied robust VRAC currents could be induced at constant cell volume by G-protein activation with GTPγS infusion. This study provides the first extensive comparative functional and pharmacological analysis of VRAC currents in normal and CF airway epithelial cells and shows that VRACs are unimpaired molecularly or functionally in CF.

Contribution of kv7.4/kv7.5 heteromers to intrinsic and calcitonin gene-related peptide-induced cerebral reactivity.

OBJECTIVE: Middle cerebral artery (MCA) diameter is regulated by inherent myogenic activity and the effect of potent vasodilators such as calcitonin gene-related peptide (CGRP). Previous studies showed that MCAs express KCNQ1, 4, and 5 potassium channel genes, and the expression products (Kv7 channels) participate in the myogenic control of MCA diameter. The present study investigated the contribution of Kv7.4 and Kv7.5 isoforms to myogenic and CGRP regulation of MCA diameter and determined whether they were affected in hypertensive animals. APPROACH AND RESULTS: Isometric tension recordings performed on MCA from normotensive rats produced CGRP vasodilations that were inhibited by the pan-Kv7 channel blocker linopirdine (P<0.01) and after transfection of arteries with siRNA against KCNQ4 (P<0.01) but not KCNQ5. However, isobaric myography revealed that myogenic constriction in response to increases in intravascular pressure (20-80 mm Hg) was affected by both KCNQ4 and KCNQ5 siRNA. Proximity ligation assay signals were equally abundant for Kv7.4/Kv7.4 or Kv7.4/Kv7.5 antibody combinations but minimal for Kv7.5/Kv7.5 antibodies or Kv7.4/7.1 combinations. In contrast to systemic arteries, Kv7 function and Kv7.4 abundance in MCA were not altered in hypertensive rats. CONCLUSIONS: This study reveals, for the first time to our knowledge, that in cerebral arteries, Kv7.4 and Kv7.5 proteins exist predominantly as a functional heterotetramer, which regulates intrinsic myogenicity and vasodilation attributed to CGRP. Surprisingly, unlike systemic arteries, Kv7 activity in MCAs is not affected by the development of hypertension, and CGRP-mediated vasodilation is well maintained. As such, cerebrovascular Kv7 channels could be amenable for therapeutic targeting in conditions such as cerebral vasospasm.

K(V)7 potassium channels: a new therapeutic target in smooth muscle disorders.

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