Synergistic interplay of Gβγ 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). βγ G proteins (Gβγ) have been identified as novel positive regulators of Kv7.4. The present study ascertained whether Gβγ increased Kv7.4 open probability through an increased sensitivity to PIP2. In HEK cells stably expressing Kv7.4, PIP2 or Gβγ increased open probability in a concentration dependent manner. Depleting PIP2 prevented any Gβγ-mediated stimulation whilst an array of Gβγ inhibitors prohibited any PIP2-induced current enhancement. A combination of PIP2 and Gβγ 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 Gβγ 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 Gβγ

G-protein βγ 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 βγ 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, Gβγ 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 Gβγ subunits, prevented these stimulatory effects. Moreover, gallein and two other structurally different Gβγ subunit inhibitors (GRK2i and a β-subunit antibody) abolished Kv7 channel currents in the absence of either Gβγ subunit enrichment or G-protein-coupled receptor stimulation. Proximity ligation assay revealed that Kv7.4 and Gβγ 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 Gβγ subunits from α subunits without stimulating nucleotide exchange, relaxed precontracted arteries in a linopirdine-sensitive manner. These results reveal that Gβγ subunits are fundamental for Kv7.4 activation and crucial for vascular Kv7 channel activity, which has major consequences for the regulation of arterial tone

TMEM16A is implicated in the regulation of coronary flow and is altered in hypertension

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

Key role for Kv11.1 (ether-a-go-go related 2 gene) channels in rat bladder contractility.

Background and purpose: In addition, to their established role in cardiac myocytes and neurons, ion channels encoded by ether-a-go-go related genes (kcnh2) are functionally relevant in phasic smooth muscle. The aim of the study was to determine the expression and functional impact of Kv11.1 in rat urinary bladder smooth muscle. Experimental approach: Quantitative polymerase chain reaction, immunocytochemistry, whole cell patch clamp, isometric tension recording. Key Results: kcnh2 was expressed in rat bladder whereas kcnh6 and 3 expression was negligible. Immunofluorescence for Kv11.1 was detected in the membrane of isolated smooth muscle cells. Potassium currents with voltage-dependent characteristics consistent with Kv11.1 channels and sensitive to the specific blocker E4031 (1μM) were recorded from isolated detrusor smooth muscles. Disabling Kv11.1 activity with specific blockers (E4031 and dofetilide, 0.2- 20μM) augmented spontaneous contractions to a greater extent than BKCa channel blockers, enhanced carbachol-driven activity, increased nerve-stimulation mediated contractions and impaired β-adrenoceptor-mediated inhibitory responses. Conclusion and implications: These data establish for the first time that Kv11.1 channels are key determinants of contractility in rat detrusor smooth muscle

Vasorelaxant effects of novel Kv7.4 channel enhancers ML213 and NS15370

BACKGROUND AND PURPOSE: The KCNQ-encoded voltage-gated potassium channel family (K(v)7.1-K(v)7.5) are established regulators of smooth muscle contractility, where K(v)7.4 and K(v)7.5 predominate. Various K(v)7.2–7.5 channel enhancers have been developed that have been shown to cause a vasorelaxation in both rodent and human blood vessels. Recently, two novel K(v)7 channel enhancers have been identified, ML213 and NS15370, that show increased potency, particularly on K(v)7.4 channels. The aim of this study was to characterize the effects of these novel enhancers in different rat blood vessels and compare them with K(v)7 enhancers (S-1, BMS204352, retigabine) described previously. We also sought to determine the binding sites of the new K(v)7 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 EC(50)s that were far lower than other K(v)7 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 K(v)7 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