LRRC8A Anion Channel Associates with Myosin Phosphatase Rho Interacting Protein (MPRIP) to Regulate Vascular Function

Abstract

Volume-regulated anion channels (VRACs) are activated by tumor necrosis factor alpha (TNFα) and angiotensin II. VRACs are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) and LRRC8A is required for all VRACs. LRRC8A associates with NADPH oxidase 1 (Nox1) and modulates production of extracellular superoxide in vascular smooth muscle cells (VSMCs). We investigated vascular reactivity in mesenteric arteries from wild type (WT) and smooth muscle-specific LRRC8A knockout (KO) mice. KO vessels displayed enhanced responsiveness to multiple vasodilator agents; acetylcholine, sodium nitroprusside, PKA (forskolin) or PKG (BAY60-2770) activators and a Rho kinase inhibitor (Y-27632), compared to WT. This was associated with decreased RhoA activity and reduced ROCK-dependent MYPT1 phosphorylation at T853. To explore the mechanism of enhanced relaxation we immunoprecipitated LRRC8A-associated proteins from WT and KO VSMCs and analyzed these by mass spectroscopy. Myosin Phosphatase Rho-Interacting protein (MPRIP) was identified only in WT lysates. MPRIP is a scaffolding protein that binds to RhoA, MYPT1, and actin and regulates activity of the myosin light chain phosphatase complex. Association of LRRC8A and MPRIP was confirmed using IP/western blot, proximity ligation assay (PLA) and immunohistochemistry. LRRC8A binds to the second plextrin homology domain of MPRIP. Knockdown of MPRIP (siRNA) increased protein expression of LRRC8A in cultured VSMCs. To explore the relationship between Nox1/LRRC8A, superoxide and intracellular redox signaling events sulfenylation of MPRIP cysteine residues (-SOH) was quantified using DCP-Bio1 (a biotin-labeled dimedone derivative) pulldown with streptavidin beads and western blotting. Sulfenylation was significantly increased 3 min following TNFα stimulation (2.3 fold, n = 3, p < 0.05). It remains to be determined how this impacts association of MPRIP with its four binding partners. Disruption of interaction between Nox1/LRRC8A and MPRIP/RhoA/MYPT1/actin may underly enhanced vasodilation in VSMC-specific LRRC8A null blood vessels. This multi-protein complex provides a potential mechanism by which oxidants produced by Nox1 impact vasomotor function, linking inflammation to increased vascular reactivity and hypertension. NIGMS (5R35GM138191-02), NIDDK (1R01DK132948-01), NHLBI (1R01HL160975-01A1) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.