Caveolae Act as Membrane Reserves Which Limit Mechanosensitive ICl,swell Channel Activation during Swelling in the Rat Ventricular Myocyte

Background: Many ion channels are preferentially located in caveolae where compartmentalisation/scaffolding with signal transduction components regulates their activity. Channels that are mechanosensitive may be additionally dependent on caveolar control of the mechanical state of the membrane. Here we test which mechanism underlies caveolar-regulation of the mechanosensitive I-Cl,I-swell channel in the adult cardiac myocyte. Methodology/Principal Findings: Rat ventricular myocytes were exposed to solution of 0.02 tonicity (T; until lysis), 0.64T for 10-15 min (swelling), and/or methyl-beta-cyclodextrin (MBCD; to disrupt caveolae). MBCD and 0.64T swelling reduced the number of caveolae visualised by electron microscopy by 75 and 50% respectively. MBCD stimulated translocation of caveolin 3 from caveolae-enriched buoyant membrane fractions, but both caveolin 1 and 3 remained in buoyant fractions after swelling. I-Cl,I-swell inhibition in control cells decreased time to half-maximal volume (t(0.5,vol); 0.64T), consistent with a role for I-Cl,I-swell in volume regulation. MBCD-treated cells showed reduced time to lysis (0.02T) and t(0.5,vol) (0.64T) compared with controls. The negative inotropic response to swelling (an index of I-Cl,I-swell activation) was enhanced by MBCD. Conclusions/Significance: These data show that disrupting caveolae removes essential membrane reserves, which speeds swelling in hyposmotic conditions, and thereby promotes activation of I-Cl,I-swell. They illustrate a general principle whereby caveolae as a membrane reserve limit increases in membrane tension during stretch/swelling thereby restricting mechanosensitive channel activation.