Several studies have linked various mechanosensitive chloride channels (MCCs) to specialized membrane structures known as caveolae ( 1, 2, 3, 4). Although all mechanosensitive chloride channels, including chloride channel 2, chloride channel 3, and SWELL-1, associate with the caveolar scaffolding protein Cav3, only the interaction between SWELL1 and Cav3 is dependent on membrane tension and caveola integrity and could be implicated in pathological remodeling of I Cl,swell observed in various cardiac pathologies conditioned by pressure and/or volume overload of the heart that are associated with caveola remodeling. Cav3 expression upregulates the SWELL1 protein expression level and its activity in HEK293 cells.

This effect is also observed in mouse ventricular myocytes. Cav3-positive HEK293 cells are highly resistant to swelling-induced membrane damage, which is reduced via I Cl,swell inhibition. Mechanoprotective properties imparted by caveola-generating caveolin-3 (Cav3) expression is facilitated by complimentary SWELL1 mechanosensitive chloride channel expression and activity via a corresponding swelling-activated chloride current ( I Cl,swell). This study highlights the mechanoprotective role of Cav3, which is facilitated by complimentary SWELL1 expression and activity. Our findings indicate that, in the MCCs tested, SWELL1 abundance and activity are regulated by Cav3 and that their association relies on membrane tension and caveola integrity. A close association between Cav3 and SWELL1 was confirmed by co-immunoprecipitation analysis. Cav3/SWELL1 membrane Förster resonance energy transfer efficiency was halved in mild (220 milliosmole) hypotonic solution as well as after disruption of caveola structures via cholesterol depletion by 1-h treatment with 10 mM methyl-β-cyclodextrin.

A higher resistance to hypotonic swelling in Cav3-positive HEK293 cells was accompanied by a significant twofold increase of I Cl,swell current density and SWELL1 protein expression, whereas ClC-2/3 protein levels remained unchanged. Förster resonance energy transfer analysis showed a less than 10-nm membrane and intracellular association between Cav3 and SWELL1. These results were recapitulated in isolated mouse ventricular myocytes, where the percentage of cardiomyocytes with membrane damage increased from 47% in control cells to 78% in 4-butanoic acid-treated cells (p < 0.05). This mechanoprotection was significantly reduced (p < 0.05) when cells were exposed to the I Cl,swell-selective inhibitor 4-butanoic acid (10 μM).

Here we showed that Cav3-transfected (Cav3-positive) HEK293 cells were significantly resistant to extreme (<20 milliosmole) hypotonic swelling compared with native (Cav3-negative) HEK293 cells the percentage of cells with membrane damage decreased from 45% in Cav3-negative cells to 17% in Cav3-positive cells (p < 0.05). However, the role of the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCC expression, activity, and contribution to membrane integrity in response to mechanical stress remains unclear. Caveola membrane structures harbor mechanosensitive chloride channels (MCCs including chloride channel 2, chloride channel 3, and SWELL1, also known as LRRC8A) that form a swelling-activated chloride current ( I Cl,swell) and play an important role in cell volume regulation and mechanoelectrical signal transduction.

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