Biology subjects

Barrese, V.

Publications and source records attributed to Barrese, V..

4 recordsLinked to original sources

bioRxiv2026

Gβγ dually regulates the M current via increased channel surface expression and PIP2 sensitivity

The M-current, generated by voltage gated KV7.2/7.3 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 (PIP2). Here, we show that coexpression of the ubiquitous subunit dimer of heterotrimeric G-proteins, G{beta}{gamma}, with KV7.2/7.3 in Xenopus laevis oocytes doubles maximal M-current. This regulation requires free, prenylated, membrane-associated G{beta}{gamma} and operates via two distinct mechanisms: 1) increasing plasma membrane (PM) channel density and 2) stabilizing KV7.2/7.3-PIP2 coupling. Conversely, G{beta}{gamma} scavengers reduce basal KV7.2/7.3 current and weaken PIP2 coupling. Proximity ligation assays confirm colocalization of G{beta}{gamma} and KV7.2/7.3 in the PM. Peptide array and AlphaFold modeling identify putative interaction sites on the channel cytoplasmic domain. Finally, the disease-causing G{beta}1 variant I80N abolished G{beta}{gamma}-induced M-current potentiation. Together, these findings establish G{beta}{gamma} as a significant physiological regulator and potential site of vulnerability in neuronal M-current function.

physiology↗

bioRxiv2025

Identification of sodium/myo-inositol transporter 1 as a major determinant of arterial contractility

BackgroundAs 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. MethodsIsometric 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. ResultsMorpholino-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 G{beta}{gamma} subunits. The SGK1 inhibitor EMD638683 prevented the raffinose-induced increase in SMIT1 and the anti-contractile effect. ConclusionToggling 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 G{beta}{gamma} subunits. This work identified SMT1-Kv7 channel complexes and SGK1 regulation as key modulators of arterial responsiveness.

cell biology↗

bioRxiv2024

CONSTITUTIVE OPENING OF THE Kv7.2 PORE ACTIVATION GATE CAUSES KCNQ2-DEVELOPMENTAL ENCEPHALOPATHY

Pathogenic variants in KCNQ2 encoding for Kv7.2 voltage-gated potassium channel subunits cause developmental encephalopathies (KCNQ2-encephalopathies), both with and without epilepsy. We herein describe the clinical, in vitro and in silico features of two encephalopathy-causing variants (A317T, L318V) in Kv7.2 affecting two consecutive residues in the S6 activation gate undergoing large structural rearrangements during pore opening. Currents through these mutant channels displayed increased density, hyperpolarizing shifts in activation gating, and insensitivity to phosphatidylinositol 4,5-bisphosphate (PIP2), a critical regulator of Kv7 channel function; all these features are consistent with a strong gain-of-function effect. An increase in single-channel open probability, with no change in membrane abundance or single-channel conductance, was responsible for the observed gain-of-function effects. All-atoms Molecular Dynamics simulations revealed that the mutations widened the inner pore gate and stabilized a constitutively open channel configuration in the closed state, with minimal effects on the open conformation. Thus, a PIP2-independent stabilization of the inner pore gate open configuration is a novel molecular pathogenetic mechanism for KCNQ2-developmental encephalopathies.

biophysics↗

bioRxiv2023

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

1IntroductionSodium 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 approachThe 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. ResultsAll SGLT2 inhibitors produced a concentration dependent relaxation (1{micro}M-100{micro}M) 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. ConclusionsSGLT2 inhibitors relax mesenteric arteries by a novel mechanism involving the release of CGRP from sensory nerves following inhibition of the Na+/H+ exchanger.

physiology↗