Caveolin-3 prevents swelling-induced membrane damage via regulation of ICl,swell activity

Abstract

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. 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. 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). This mechanoprotection was significantly reduced (p < 0.05) when cells were exposed to the I_Cl,swell-selective inhibitor 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (10 μM). 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-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid-treated cells (p < 0.05). 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. 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 close association between Cav3 and SWELL1 was confirmed by co-immunoprecipitation analysis. 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. This study highlights the mechanoprotective role of Cav3, which is facilitated by complimentary SWELL1 expression and activity.

Figure 1

Effect of Cav3 expression and DCPIB on membrane damage and relative cell size during hypotonic stress. (A) Representative images of Cav3-negative (Cav3(−)) HEK293 cells in isotonic and extreme hypotonic solutions with membrane damage (blebbing or lysis) or undergoing a regulatory volume decrease. (B) Percentage of Cav3(−) and Cav3-positive (Cav3(+)) HEK293 cells with membrane damage, treated with 10 μM DCPIB and incubated for 15 min in extreme hypotonic solution. Cav3(+) HEK293 cells were also treated with or without DCPIB and isotonic solution as a negative control. (C) Relative minimum and maximum cell size of Cav3(−) and Cav3(+) HEK293 cells treated with 10 μM DCPIB HEK-293 or left untreated under isotonic and extreme hypotonic conditions. N = 3 groups, n = 3 samples/group. (D and E) Cav3(−) and Cav3(+) HEK293 cell relative cell volume with extreme hypotonic incubation (Hypo) or extreme hypotonic incubation with 10 μM DCPIB (Hypo + DCPIB) over 15 min ^∗p < 0.05 compared with time 0. N = 4–5 groups, n > 5 cells/group. p Values within cell types were calculated with paired Student’s t-tests. p Values between cell types were calculated with one-way ANOVA. Mean ± SE.

Figure 2

Effect of DCPIB and MβCD on swelling-induced membrane damage and cell width in cardiomyocytes. (A) Representative images of isolated mouse ventricular myocytes in isotonic (1 T) and highly hypotonic solutions (0.1 T) with membrane damage (blebbing or lysis) or no damage. (B) Percentage of isolated mouse ventricular myocytes with membrane damage, treated with 10 μM DCPIB and/or 10 mM MβCD and incubated for 20 min in highly hypotonic solution. ^∗p < 0.05 compared with control (0.1 T). (C) Relative maximum width of isolated mouse ventricular myocytes treated with 10 μM DCPIB and/or 10 mM MβCD under highly hypotonic conditions. (D and E) Relative cell width of isolated mouse ventricular myocytes incubated with high hypotonic solution supplemented with 10 μM DCPIB and/or 10 mM MβCD over 12 min. ^∗p < 0.05 compared with time 0, ##p < 0.05 between the same time points. N = 3–5 groups, n = 3–10 cells/group. Membrane damage p values were calculated using a one-way ANOVA Kruskal-Wallis test. All other p values were determined via one-way ANOVA. Mean ± SE.

Figure 3

Cav3 expression increases caveolae abundance in HEK293 cells. (A) Representative image of Cav3(+) HEK293 cells with closed and open caveolae. (B) Average number of open, closed, and total caveolae per cell in Cav3(−) and Cav3(+) HEK293 cells. N = 4 samples, n = 30 cells/sample. (C) Average relative Cav3(−) and Cav3(+) HEK293 cell perimeter in arbitrary units. N = 4 samples, n = 10–20 cells/sample. (D) Average number of total caveolae per cell in Cav3(−) and Cav3(+) HEK293 cells, corrected by average relative cell perimeter. N = 4 samples, n = 30 cells/sample. Mean ± SE. Caveolae number p values were calculated with unpaired Student’s t-test. Relative cell perimeter p value was determined via Welch’s t test. Mean ± SE.

Figure 4

CBMs for MCCs. (A) Structure of the caveola scaffolding protein caveolin-3. Caveolin-3 has four primary domains: NH2-terminal domains; scaffolding domains that form α-helices and are inserted into the membrane, with a cholesterol recognition/interaction amino acid consensus composed of the eight residues proximal to the membrane domain; helix-turn-helix membrane domains; and COOH-terminal domains. Bars indicate separation of protein domains. From Busija et al. (75) with permission. (B–D) Location of possible CBMs (shown by asterisks) within SWELL1, ClC-2, and ClC-3 protein structures. CBS, cystathionine β synthase. (E) CBM counts relative to non-caveola-associated proteins.

Figure 5

Effect of Cav3 on MCC protein expression in HEK293 cells. (A) SWELL1 antibody (rabbit polyclonal; SAB2108060, Sigma-Aldrich) validation, showing representative Western blots of SWELL1 with/without siRNA treatment; other antibodies have been validated previously (2). (B) Quantification of SWELL1 siRNA relative to GAPDH. (C) Representative Western blots of MCCs, GAPDH, and Cav3 in Cav3(−) and Cav3(+) HEK293 cell lines. (D) qPCR SWELL1 CT quantification relative to GAPDH with and without Cav3 expression. (E1–E3) Quantification of MCC protein expression with and without Cav3 expression. (F) Co-immunoprecipitation of SWELL1 and Cav3 in Cav3(+) HEK293 cell lines. IP-A, immunoprecipitation via 2.5% acetic acid; IP-S, immunoprecipitation via sodium dodecyl sulfate; FT, flowthrough. N = 3 groups, n = 3–7 samples/group. Mean ± SD. p Values were determined by unpaired Student’s t-tests. n.s., non-significant.

Figure 6

Cav3 increases I_Cl,swell density in HEK293 cells. (A) Representative current traces illustrating I_Cl,swell currents in isotonic (Iso), hypotonic (Hypo), and Hypo with 10 μM DCPIB (Hypo + DCPIB) solutions in Cav3(−) (wild-type) and Cav3(+) HEK293 cells. Whole-cell currents were recorded using the protocol indicated in the inset. (B–D) Mean current-voltage relationships of peak I_Cl,swell density in Iso, Hypo, and Hypo + DCPIB solution in Cav3(−) HEK293 cells (B) (n = 5–8 cells; ^∗∗∗,^∗p < 0.001, 0.05 between Hypo and Iso; ###,##p < 0.001, 0.01 between Hypo and Hypo + DCPIB by one-way ANOVA with Bonferroni correction) and Cav3(+) HEK293 cells (C) (n = 4–9 cells; ^∗∗∗,^∗∗p < 0.001, 0.01 between Hypo and Iso; ###,##,#p < 0.001, 0.01, 0.05 between Hypo and Hypo + DCPIB by one-way ANOVA with Bonferroni correction) as well as a comparison between hypotonically activated I_Cl,swell in Cav3(−) versus Cav3(+) HEK-293 cells (D) ($$,$p < 0.01, 0.05 between Cav3(+) and Cav3(−) (wild-type) groups by one-way ANOVA with Bonferroni correction).

Figure 7

Representative images and FRET of HEK293 cells transfected with the pCAG-GFP, Cav3-GFP, and SWELL1-mCherry plasmids. (A) pCAG-GFP and SWELL1-mCherry transfections. (B) Cav3-GFP and SWELL1-mCherry transfections.

Figure 8

Representative images and FRET of HEK293 cells transfected with the pCAG-GFP, Cav3-GFP, ClC-2-mCherry, and ClC-3-mCherry plasmids. (A) pCAG-GFP and ClC-2-mCherry transfections. (B) pCAG-GFP and ClC-3-mCherry transfections. (C) Cav3-GFP and ClC-2-mCherry transfections. (D) Cav3-GFP and ClC-3-mCherry transfections.

Figure 9

Membrane colocalization of Cav3-GFP, pCAG-GFP, SWELL1-mCherry, ClC-2-mCherry, and ClC-3 mCherry. (A) Representative image of Cav3-GFP and SWELL1-mCherry membrane colocalization. (B-D) Mander’s coefficients of Cav3-GFP/SWELL1-mCherry and pCAG-GFP/SWELL1-mCherry (B), Cav3-GFP/ClC-2-mCHerry and pCAG-GFP/ClC-2-mCherry (C), and Cav3-GFP/ClC-mCherry and pCAG-GFP/ClC-3-mCherry (D).

Figure 10

Cav3/MCC and pCAG/MCC FRET quantification under Iso (1 T), Hypo (0.7 T), and MβCD conditions. (A–C) Summarized FRET results for the membrane Cav3/MCC FRET (A1, B1, and C1), membrane control pCAG/SWELL1 (A2, B2, and C2), and intracellular Cav3/SWELL1 FRET (A3). FRET quantifications for baseline (Iso, 1 T) and after 1-h incubation with 10 mM MβCD Iso conditions, alone or together with 6.5 μM cholesterol for membrane Cav3/SWELL1 FRET (D1), cholesterol control Cav3/SWELL1 FRET (D2), and intracellular Cav3/SWELL1 FRET (D3). N = 9 cells/group, 10–15 FRET signals collected/cell, N = 3 transfections. Mean ± SD. p Values determined by paired Student’s t-tests.