Abstract P368: Caveolin-3 Prevents Swelling-induced Cell Lysis Via Regulation Of I _Cl,swell Expression And Activity

Background: Caveolae membrane structures harbor mechanosensitive chloride channels (MCCs) which form a swelling-activated chloride current ( I Cl,swell ) and play an important role in cell volume regulation and mechano-electrical signal transduction. However, the role of muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCCs expression, activity, and contribution to cell viability in response to mechanical stress remains unclear. We hypothesized that Cav3-based mechano-protection is enabled by complimentary expression of MCCs. Methods and Results: Experiments were performed on native (Cav3-) and Cav3-transfected (Cav3+) HEK-293 cells. Cell stretch was mimicked by light (220 mOsM) or extreme hypoosmotic swelling (<20mOsM). Cav3+ HEK-293 cells were significantly resistant to extreme hypotonic solutions (15 minute incubation) compared to Cav3- HEK-293 cells, and this mechano-protection was significantly reduced when exposed to I Cl,swell selective inhibitor DCPIB (1 μM). We found that three MCCs (ClC-2, ClC-3, and SWELL1, also known as LRRC8A) contain caveolin-binding motifs in their structure, indicating their possible localization in caveolae structures. Co-IP analysis confirmed association of SWELL1 with Cav3. Interestingly, Cav3+ HEK-293 cells showed a significant (by 2-fold) increase of SWELL1 protein level, while ClC-2/3 protein levels remained unchanged. This was accompanied by a 2-fold increase of I Cl,swell , but no change in mRNA expression levels. FRET analysis showed a <10 nm membrane and intracellular association between Cav3 and tested MCCs. Furthermore, Cav3/SWELL1 membrane FRET efficiency was halved in light hypoosmotic solution, as well as after disruption of caveolae structures via cholesterol depletion by 1-hour treatment with 10 μM methyl-β-cyclodextrin. Cav3/ClC-2/3 average membrane FRET efficiency remained unchanged in hypotonic solution. Conclusions: We concluded that of MCCs tested, SWELL1 abundance and activity is regulated by Cav3 and that their association relies on membrane tension and caveolae integrity. The present study highlights the mechano-protective properties of Cav3 which are partially facilitated by complimentary SWELL1 expression and activity.