Disruption of Lipid Rafts Inhibits P2X1 Receptor-mediated Currents and Arterial Vasoconstriction

Abstract

P2X1 receptors for ATP are ligand-gated cation channels expressed on a range of smooth muscle preparations and blood platelets. The receptors appear to be clustered close to sympathetic nerve varicosities and mediate the underlying membrane potential changes and constriction following nerve stimulation in a range of arteries and resistance arterioles. In this study we have used discontinuous sucrose density gradients, Western blot analysis, and cholesterol measurements to show that recombinant and smooth muscle (rat tail artery, vas deferens, and bladder) P2X1 receptors are present in cholesterol-rich lipid rafts and co-localize with the lipid raft markers flotillin-1 and -2. Lipid rafts are specialized lipid membrane microdomains involved in signaling and trafficking. To determine whether lipid raft association was essential for P2X1 receptor channel function we used the cholesterol-depleting agent methyl-beta-cyclodextrin (10 mm for 1 h). This led to a redistribution of the P2X1 receptor throughout the sucrose gradient and reduced P2X1 receptor-mediated (alpha,beta-methylene ATP, 10 microm) currents in HEK293 cells by >90% and contractions of the rat tail artery by approximately 50%. However contractions evoked by potassium chloride (60 mm) were unaffected by methyl-beta-cyclodextrin and the inactive analogue alpha-cyclodextrin had no effect on P2X1 receptor-mediated currents or contractions. P2X1 receptors are subject to ongoing regulation by receptors and kinases, and the present results suggest that lipid rafts are an essential component in the maintenance of these localized signaling domains and play an important role in P2X1 receptor-mediated control of arteries.