Iain Andrew Greenwood

The M-current, generated by voltage gated K _V 7.2/7.3 channels, sets the threshold for neuronal action potential and acts as a key brake on repetitive firing. The M-current is tightly regulated by signaling molecules, such as calmodulin and phosphatidylinositol-4,5-bisphosphate (PIP ₂ ). Here, we show that coexpression of the ubiquitous subunit dimer of heterotrimeric G-proteins, Gβγ, with K _V 7.2/7.3 in Xenopus laevis oocytes doubles maximal M-current. This regulation requires free, prenylated, membrane–associated Gβγ and operates via two distinct mechanisms: 1) increasing plasma membrane (PM) channel density and 2) stabilizing K _V 7.2/7.3-PIP₂ coupling. Conversely, Gβγ scavengers reduce basal K _V 7.2/7.3 current and weaken PIP ₂ coupling. Proximity ligation assays confirm colocalization of Gβγ and K _V 7.2/7.3 in the PM. Peptide array and AlphaFold modeling identify putative interaction sites on the channel’s cytoplasmic domain. Finally, the disease–causing Gβ ₁ variant I80N abolished Gβγ–induced M-current potentiation. Together, these findings establish Gβγ as a significant physiological regulator and potential site of vulnerability in neuronal M-current function.

KV7 (KCNQ) channels generate slowly activating, non-inactivating K+ currents that stabilize the membrane potential and limit cellular excitability [1–3]. Within the cardiovascular and visceral syst...

Background: As the sodium/ myoinositol transporter (SMIT1) is a positive regulator of Kv7.4/7.5 channels in arterial smooth muscle we postulated that altering SMIT1 expression could have a major impact upon vascular reactivity. Consequently, this study aimed to characterise the effects of changes of SMIT1 membrane abundance on vascular tone and the molecular mechanisms involved. Methods: Isometric tension recording on 2nd order mesenteric arteries and left anterior coronary arteries from male and female Wistar Han rats. Whole artery membrane potential recording. Single cell and whole artery antibody-based imaging. Morpholino based protein knockdown of SMIT1. Results: Morpholino-mediated knockdown of SMIT1 enhanced U46619- and methoxamine-mediated contractions of mesenteric artery whilst impairing relaxations to the Kv7 activator ML213, isoprenaline and CGRP. Conversely, augmenting SMIT1 membrane abundance by raising external osmolarity with 150 mM raffinose, impaired receptor-mediated contractions of mesenteric and coronary arteries, augmented relaxations to ML213 and adenosine as well as producing membrane potential hyperpolarisation. Proximity ligation assays revealed that raffinose incubation increased the association of SMIT-Kv7.4/7.5 as well as Kv7.4 and Gβγ subunits. The SGK1 inhibitor EMD638683 prevented the raffinose-induced increase in SMIT1 and the anti-contractile effect. Conclusion: Toggling the membrane abundance of SMIT1 had a dramatic effect on the arterial response to vasonstrictors and vasodilators mediated by greater coordination of Kv7 channels and Gβγ subunits. This work identified SMT1-Kv7 channel complexes and SGK1 regulation as key modulators of arterial responsiveness.

Despite spontaneous preterm birth (PTB, delivery < 37 weeks’ gestation) being a major contributor to perinatal morbidity and mortality worldwide, there is a paucity of treatments for treating preterm labor. We hypothesized that uterine smooth muscle (myometrium) Kv7 channels could be a therapeutic target for preventing preterm labor. Building on our previous work, we confirmed that K _V 7 channels, proteins encoded by KCNQ2-5 genes and associated accessory KCNE1-5, are expressed and functional in pregnant human myometrium prior to and after the onset of labor. K _V 7.2-5 activators (retigabine and ML213) effectively inhibit pregnant human and mouse myometrium contractions in vitro , and in vivo significantly delayed PTB in a non-infection preterm labor mouse model. This supports our hypothesis that augmenting K _V 7 activity represents a viable mechanism to suppress uterine contractility and delay of PTB. Addressing the current drive to repurpose existing drugs for treating PTB, we propose this as a new avenue of clinical exploration. One Sentence Summary : K _V 7 channel activators inhibit human uterine contractions and delay delivery in preterm mice, identifying a new target for preterm labor prevention.

This study investigated the sex and oestrous‐stage dependent effects on voltage‐gated potassium channels encoded by KCNQ genes (Kv7) channels in the rat bladder, focusing on the roles of different estrogen receptors (ERs). We hypothesized that ERα and G‐protein‐coupled estrogen receptor (GPER1) play distinct roles in modulating Kv7.4 and Kv7.5 channel functionality and localization. Using bladder tissues from male and female Wistar rats at different oestrous stages, we performed myography, RT‐qPCR, immunocytochemistry, and western blot analyses. Our results showed that ML213, a Kv7.2–7.5 activator, was more effective in male bladders, correlating with higher membrane localization of Kv7.4 and Kv7.5. In contrast, female rats in the proestrous and oestrous stages exhibited reduced sensitivity to ML213, associated with decreased Kv7.5 membrane abundance. In this group, ERα attenuated both the Kv7 functional response to ML213 and the membrane localization of Kv7.5, whereas GPER1 was associated with reducing the membrane abundance of the channel without affecting functionality. In diestrous and metestrous rat bladders, ERα was not active in baseline conditions. Instead, GPER1 induced a removal of Kv7.5 from the membrane, without exerting any effect on the Kv7 functional response to ML213. These findings highlight a complex interplay between sex hormones and ERs in bladder smooth muscle cells, offering insights into sex‐specific differences in bladder function and potential therapeutic targets for bladder disorders.

Aims: Sodium/glucose transporter 2 (SGLT2 or SLC5A2) inhibitors lower blood glucose and are also approved treatments for heart failure independent of raised glucose. Various studies have showed that SGLT2 inhibitors relax arteries, but the underlying mechanisms are poorly understood and responses variable across arterial beds. We speculated that SGLT2 inhibitor-mediated arterial relaxation is dependent upon calcitonin gene-related peptide (CGRP) released from sensory nerves independent of glucose transport. Methods and results: The functional effects of SGLT1 and 2 inhibitors (mizagliflozin, dapagliflozin, and empagliflozin) and the sodium/hydrogen exchanger 1 (NHE1) blocker cariporide were determined on pre-contracted resistance arteries (mesenteric and cardiac septal arteries) as well as main renal conduit arteries from male Wistar rats using wire myography. SGLT2, CGRP, TRPV1, and NHE1 expression 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. All SGLT inhibitors (1–100 µM) and cariporide (30 µM) relaxed mesenteric arteries but had negligible effect on renal or septal arteries. Immunohistochemistry with TRPV1 and CGRP antibodies revealed a dense innervation of sensory nerves in mesenteric arteries that were absent in renal and septal arteries. Consistent with a greater sensory nerve component, the TRPV1 agonist capsaicin relaxed mesenteric arteries more effectively than renal or septal arteries. In mesenteric arteries, relaxations to dapagliflozin, empagliflozin, and cariporide were attenuated by the CGRP receptor antagonist BIBN-4096, depletion of sensory nerves with capsaicin, and blockade of TRPV1 or Kv7 channels. Neither dapagliflozin nor empagliflozin activated heterologously expressed TRPV1 channels or Kv7 channels directly. Sensory nerves also expressed NHE1 but not SGLT2 and cariporide pre-application as well as knockdown of NHE1 by translation stop morpholinos prevented the relaxant response to SGLT2 inhibitors. Conclusion: SGLT2 inhibitors relax mesenteric arteries by promoting the release of CGRP from sensory nerves in a NHE1-dependent manner.

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 βγ subunits (Gβγ) 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.

1 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.

Pulmonary arterial (PA) smooth muscle cells (PASMC) generate vascular tone in response to agonists coupled to G_q-protein receptor signaling. Such agonists stimulate oscillating calcium waves, the frequency of which drives the strength of contraction. These Ca²⁺ events are modulated by a variety of ion channels including voltage-gated calcium channels (Ca_V1.2), the Tmem16a or Anoctamin-1 (ANO1)-encoded calcium-activated chloride (CaCC) channel, and Ca²⁺ release from the sarcoplasmic reticulum through inositol-trisphosphate receptors (IP₃R). Although these calcium events have been characterized, it is unclear how these calcium oscillations underly a sustained contraction in these muscle cells. We used smooth muscle–specific ablation of ANO1 and pharmacological tools to establish the role of ANO1, Ca_V1.2, and IP₃R in the contractile and intracellular Ca²⁺ signaling properties of mouse PA smooth muscle expressing the Ca²⁺ biosensor GCaMP3 or GCaMP6. Pharmacological block or genetic ablation of ANO1 or inhibition of Ca_V1.2 or IP₃R, or Ca²⁺ store depletion equally inhibited 5-HT-induced tone and intracellular Ca²⁺ waves. Coimmunoprecipitation experiments showed that an anti-ANO1 antibody was able to pull down both Ca_V1.2 and IP₃R. Confocal and superresolution nanomicroscopy showed that ANO1 coassembles with both Ca_V1.2 and IP₃R at or near the plasma membrane of PASMC from wild-type mice. We conclude that the stable 5-HT-induced PA contraction results from the integration of stochastic and localized Ca²⁺ events supported by a microenvironment comprising ANO1, Ca_V1.2, and IP₃R. In this model, ANO1 and Ca_V1.2 would indirectly support cyclical Ca²⁺ release events from IP₃R and propagation of intracellular Ca²⁺ waves.

In addition, to their established role in cardiac myocytes and neurons, ion channels encoded by ether‐a‐go‐go‐related genes (ERG1‐3 or kcnh2,3 and 6) (kcnh2) are functionally relevant in phasic smooth muscle. The aim of the study was to determine the expression and functional impact of ERG expression products in rat urinary bladder smooth muscle using quantitative polymerase chain reaction, immunocytochemistry, whole‐cell patch‐clamp and isometric tension recording. kcnh2 was expressed in rat bladder, whereas kcnh6 and kcnh3 expression were negligible. Immunofluorescence for the kcnh2 expression product 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 (1 μM) were recorded from isolated detrusor smooth muscles. Disabling Kv11.1 activity with specific blockers (E4031 and dofetilide, 0.2–20 μM) augmented spontaneous contractions to a greater extent than BK_Ca channel blockers, enhanced carbachol‐driven activity, increased nerve stimulation‐mediated contractions, and impaired β‐adrenoceptor‐mediated inhibitory responses. These data establish for the first time that Kv11.1 channels are key determinants of contractility in rat detrusor smooth muscle.