Impairment of TRPC1-STIM1 channel assembly and AQP5 translocation compromise agonist-stimulated fluid secretion in mice lacking caveolin1

Abstract

Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca(2+) influx. The Ca(2+)-permeable transient receptor potential canonical 1 (TRPC1) channel is crucial for fluid secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is essential for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo and thus regulates fluid secretion. In Cav1(-/-) mouse SG, agonist-stimulated Ca(2+) entry and fluid secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1(-/-) SG acinar cells, whereas Orai1-STIM1 interaction is not affected. Furthermore, localization of aquaporin 5 (AQP5), but not that of inositol (1,4,5)-trisphosphate receptor 3 or Ca(2+)-activated K(+) channel (IK) in the apical region of acinar cell was altered in Cav1(-/-) SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca(2+) influx via TRPC1 channels and was inhibited in Cav1(-/-) SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1(-/-) SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1-STIM1 channels, Cav1 is a crucial determinant of SG fluid secretion.

Fig. 1.

Loss of caveolae abrogates TRPC1 localization. (A) TEM images of acinar cell regions (caveolar microdomains shown by arrows). BLM, basolateral membrane; A, acini. Scale bars: 250 nm. (B) Confocal images of SMG sections showing TRPC1 (with DAPI) and STIM1 staining in Cav1^+/+ and Cav1^−/− acini. Scale bars: 10 µm. (C) Western blots performed on density gradient fractions of SMGs showing raft and non-raft distributions of proteins. Blots presented are representative of 3–4 independent fractionations. (D) Western blots showing basal (Ctrl, control) and Pilocarpine-stimulated (Pilo) distribution of proteins in raft and non-raft fractions (Di). BF, raft or buoyant fractions 3–4; HF, non-raft or heavy fractions 8–10. Bar graph (Dii) summarizes densitometric analysis of STIM1 from four independent experiments. Values indicate mean ± s.d.; *P<0.05, **P<0.01.

Fig. 2.

Cav1 ablation attenuates TRPC1-STIM1 interaction and function. (A) Co-immunoprecipitation in resting (Ctrl, control) and pilocarpine-stimulated (Pilo) SMGs from Cav1^+/+ (Ai) and Cav1^−/− (Aii) mice. Blots presented are representative of 4–6 independent experiments. (B) Fura2 fluorescence traces (average of at least 30–40 traces) of Tg-induced [Ca²⁺]_i changes (Bi); bar graph (Bii) shows quantifications of Tg-induced Ca²⁺ release and influx and CCh-induced Ca²⁺ influx. (C) Tg-induced I-SOC measurements of currents (Ci) and IV curves (Cii) in dispersed acinar cells of Cav1^+/+ and Cav1^−/− SMGs; bar graph (Ciii) shows quantification of the current density. Numbers enclosed in parentheses indicate traces analyzed. Values indicate mean ± s.d.; **P<0.01.

Fig. 3.

Cav1 is essential for agonist-induced salivary fluid secretion. (A) Pilocarpine-stimulated cumulative whole-saliva secretions measured from Cav1^+/+ and Cav1^−/− animals over 10 minutes. (B) Bar graph quantifying the total volume (µl) of saliva secreted in 10 minutes. (C) Flow rate of saliva secretions. (D) Expression levels of proteins involved in receptor-stimulated ion regulation in SMG. Actin is used as loading control. Blots are compiled from at least 2–4 independent analysis. Values indicate mean ± s.d.; *P<0.05; n is the number of animals used in each group.

Fig. 4.

Cav1 and TRPC1 are required for AQP5 translocation. (A) Density gradient fractions showing raft and non-raft distribution of AQP5 in Cav1^+/+ and Cav1^−/− SMGs (Ai); bar graph (Aii) shows densitometric quantification of lipid raft-associated AQP5 (n = 3–4 independent fractionations). (B) Confocal images of pilocarpine-stimulated SMG sections stained for AQP5 (red), Na-K-ATPase (green) and nucleus (DAPI, blue) in Cav1^+/+ and Cav1^−/− acini (arrows point to apical regions). Scale bars: 10 µm. (C) Western blots showing cell surface biotinylation of basal (Ctrl, control) and pilocarpine-stimulated (Pilo) primary SMG acinar cells. Data presented are representative of two independent experiments. (D) Cell surface biotinylation showing CCh-stimulated (100 µM, 5 minutes) AQP5 surface expression in HSG cells with or without Cav1 or TRPC1 knockdown (Di); bar graph (Dii) summarizes quantifications from four independent experiments. Values indicate mean ± s.d.; *P<0.05.

Fig. 5.

In vivo Cav1 reconstitution restores fluid secretion. (A) TEM showing acinar cell regions of AdGFP or AdCav1 expressing Cav1^−/− SMGs. Caveolar vesicles are indicated by arrows. Scale bars: 200 nm (see supplementary material Fig. S3C,D for efficient gene delivery in the salivary glands). (B) Co-immunoprecipitation of STIM1-TRPC1-Orai1 in resting (Ctrl, control) and pilocarpine-stimulated (Pilo) Cav1^−/− SMGs expressing Cav1. Blots presented are representative of 3–4 independent experiments. (C) Tg-induced current measurements (Ci) and IV curves (Cii) in primary acinar cells; bar graph (Ciii) shows quantifications. Numbers enclosed in parentheses indicate the number of traces analyzed. (D) Fura2 fluorescence traces of CCh (100 µm)- or Tg (2 µM)-induced [Ca²⁺]_i changes (Di); bar graph (Dii) shows respective quantifications. (E) Confocal images of pilocarpine-stimulated SMG sections stained for AQP5 (red), Na-K-ATPase (green) and nucleus (DAPI, blue) in Cav1 expressing Cav1^−/− and Cav1^−/− acini (arrows point to apical regions). Scale bars: 10 µm. (F) Pilocarpine-stimulated cumulative whole-saliva secretions measured over 10 minutes in Cav1^−/− animals expressing GFP or Cav1 (Fi); bar graph (Fii) quantifies the total volume (µl) of saliva secreted in 10 minutes. Numbers in parentheses indicate the number of animals used in each group. Values indicate mean ± s.d.; *P<0.05, **P<0.01.