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Abstract
BackgroundMany 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,swell channel in the adult cardiac myocyte.Methodology/Principal FindingsRat ventricular myocytes were exposed to solution of 0.02 tonicity (T; until lysis), 0.64T for 10–15 min (swelling), and/or methyl-β-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,swell inhibition in control cells decreased time to half-maximal volume (t 0.5,vol; 0.64T), consistent with a role for I Cl,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,swell activation) was enhanced by MBCD.Conclusions/SignificanceThese data show that disrupting caveolae removes essential membrane reserves, which speeds swelling in hyposmotic conditions, and thereby promotes activation of I Cl,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.
Highlights
Caveolae are small (50–100 nm) invaginations of the plasma membrane, found in almost all cells of the body
In cells exposed to isotonic solution, both invaginations and closed subsarcolemmal vesicles are clearly evident with a diameter consistent with that of caveolae (50– 100 nm)
We have previously shown that 10 min exposure to hypotonic solution abbreviates the action potential duration in the rat ventricular myocyte, an effect sensitive to 4,49diisothiocyanostilbene-2,29-disulphonic acid (DIDS), confirming a contribution from ICl,swell [29]
Summary
Caveolae are small (50–100 nm) invaginations of the plasma membrane, found in almost all cells of the body They represent a specialised form of lipid raft, characterised by the presence of the small protein caveolin, which inserts into the inner leaflet of the membrane via a hairpin loop [1]. Cav acts as a regulator of protein activity and as a scaffold by interaction via its 20 residue scaffolding domain [4,5]. In addition to their ability to compartmentalise signalling, another property of caveolae, as a reserve of extra membrane [6], may be relevant to their functional role. We test which mechanism underlies caveolar-regulation of the mechanosensitive ICl,swell channel in the adult cardiac myocyte
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