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First published online 10 June 2008
doi: 10.1242/jcs.032003

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TRPC1 binds to caveolin-3 and is regulated by Src kinase – role in Duchenne muscular dystrophy

¹ School of Medical Sciences, Discipline of Physiology (F13), Bosch Institute, The University of Sydney, NSW 2006, Australia
² Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

View larger version (33K): [in this window] [in a new window]   Fig. 1. Levels of caveolin-3, TRPC1 and Src are increased in mdx muscle. (A) Sample immunoblots of TA muscle from mdx and WT mice. Membranes were probed for caveolin-3, TRPC1 and Src. (B-D) Total lysate (20 µg; Bradford assay) was resolved by SDS-PAGE. Bands were quantified and normalised to GAPDH (see Materials and Methods). Quantitation revealed that levels of caveolin-3, TRPC1 and Src were all increased in the mdx group. n=8 mice per group. *P<0.05; ***P<0.001.

View larger version (83K): [in this window] [in a new window]   Fig. 2. Sarcolemma levels of TRPC1 and caveolin-3 are increased in mdx muscle. (A-D) Sample micrographs of muscle cryosections from WT and mdx mice double stained by immunofluorescence for TRPC1 and caveolin-3. Scale bar: 50 µm. (E,F) Quantitation of the fluorescence in the sarcolemma region revealed increased intensity of staining for both TRPC1 and caveolin-3 in the mdx group. n=4 mice per group, 15 membrane segments per mouse. *P<0.05; ***P<0.001.

View larger version (41K): [in this window] [in a new window]   Fig. 3. TRPC1 colocalises and co-immunoprecipitates with caveolin-3. (A-H) Skeletal-muscle cryosections (TA) from mdx and WT mice were double labelled for TRPC1 (red) and caveolin-3 (green) (A-F). Images were then overlaid (C,F) and the colocalisation was quantified. (D-F) Higher-magnification images of the insets shown in A-C. Scale bars: A, 50 µm; D, 5 µm. (G) White pixels represent true colocalisation and were compared to the binary images generated from each labelling (data not shown; see Materials and Methods). Staining of caveolin and TRPC1 showed substantial but not complete overlap. (H) More than 50% of TRPC1-stained pixels colocalised with caveolin-3 staining in the sarcolemma and subsarcolemmal compartment, and vice-versa, in both mdx and WT groups. In addition, a subset of TRPC1 staining appeared to be associated with punctate subsarcolemmal structures that stained weakly with anti-caveolin3. Mean ± s.e.m. of ten membrane segments per mouse is represented, from three mice per group. (I-K) Co-immunoprecipitation of TRPC1 and caveolin-3. Total lysate from mdx and WT skeletal muscle was pre-cleared with agarose beads and incubated with anti-TRPC1. Immunocomplex was resolved by a SDS-PAGE and membranes were probed for caveolin-3. (I,J) Caveolin-3 co-immunoprecipitates with TRPC1 in both mdx and WT skeletal muscles. (K) A reverse IP also confirmed that caveolin-3 and TRPC1 co-immunoprecipitate. (I) As negative controls, samples were processed without the primary antibody (anti-TRPC1) or without the lysate. Lysate of a WT mouse was used for the IP.

View larger version (54K): [in this window] [in a new window]   Fig. 4. Caveolin-3 binds to TRPC1 and regulates its plasma membrane localisation. (A) C2 cells were transfected with either TRPC1-CFP, caveolin-3–YFP or the combination of both plasmids. Caveolin-3–YFP targeted to the plasma membrane when transfected either alone or with TRPC1. However, when TRPC1-CFP was transfected alone, it did not target to the plasma membrane and rather showed a diffuse cytoplasmic distribution. TRPC1 was only able to target to the membrane when co-transfected with caveolin-3–YFP (arrows). (B) TRPC1 binds to caveolin-3 (FRET – photobleaching of acceptor). TRPC1-CFP and caveolin-3–YFP were co-transfected into C2 myoblasts. A selected region of caveolin-3–YFP was chosen and photobleached using the 514 nm laser line (100% power). CFP fluorescence increased 21% (FRET efficiency) upon YFP photobleaching. Scale bars: A, 10 µm; B, 5 µm.

View larger version (24K): [in this window] [in a new window]   Fig. 5. CFP lifetime of TRPC1-CFP decreases when co-transfected with caveolin-3–YFP into C2 myoblasts. (A) C2 myoblasts were transfected with TRPC1-CFP alone or in combination with caveolin-3–YFP. A reduction in the CFP lifetime was detected when both plasmids were co-transfected, compared with cells transfected only with TRPC-CFP [from 2.7±0.03 nanoseconds (ns) to 2.1±0.03 ns; FRET efficiency 20%; see B,C]. Scale bar: 10 µm. (B) Example of a lifetime measurement showing the shift of the graph to the left (shortening of lifetime) in cells co-transfected with both TRPC-CPF and caveolin-3–YFP. (C) Mean of lifetime from cells transfected with TRPC-CFP only or in combination with caveolin-3–YFP. Dots represent individual cells from three independent experiments. (D) When cells co-transfected with TRPC1-CFP and caveolin-3–YFP were incubated with H2O2, an increase in the CFP lifetime was detected (from 2.17 to 2.33±0.03 ns) compared with control cells (no H2O2). This increase was prevented by incubation of the cells with PP2 prior to H2O2 treatment, which shows that Src activation can induce TRPC1 conformation changes or interfere with the binding properties involved in the interaction between TRPC1 and caveolin-3 (see Discussion). Graph represents mean ± s.e.m. of a minimum of six cells per group per experiment, from three independent experiments. ***P<0.001.

View larger version (37K): [in this window] [in a new window]   Fig. 6. H2O2 induces Src-kinase activation. C2 myoblasts were incubated with 10 µm H2O2 for 15 minutes. Immunoblots were probed for pY418-Src, striped then re-probed for total Src. A sample blot is shown. The graph represents the fold change in Src activation compared with control (pY418-Src/total Src). H2O2 incubation increased Src phosphorylation/activation 1.8 times compared with control cells. Such an increase was completely blocked when cells were incubated with PP2 prior to H2O2. Graph represents mean ± s.e.m. of four dishes per group from two independent experiments. *P<0.05; ***P<0.001.

View larger version (16K): [in this window] [in a new window]   Fig. 7. TRPC1 activity depends on caveolin-3 expression and Src phosphorylation. C2 myoblasts were transfected with TRPC1-CFP alone or in combination with caveolin-3–YFP. Cells were loaded with Fura Red and imaged using scanning confocal microscopy. During imaging, cells were incubated with H2O2 and changes in Ca2+ levels were measured. (A) Fura Red is excited using the 458 nm and 488 nm laser lines (upper graph). Relative Ca2+ concentration is represented by the ratio of Fura Red emission upon 458 nm and 488 nm excitations (ratio=Em458nm/Em488nm; lower graph). (B) C2 myoblasts were co-transfected with TRPC1-CFP and caveolin-3–YFP, loaded with Fura Red, and imaged. Increase in Ca2+ levels, compared with control (non-transfected cells; thick black line), was detected in such cells upon H2O2 incubation and this increase was prevented by PP2 incubation prior to H2O2 treatment (dotted line). In cells expressing TRPC1-CFP only (no caveolin-3–YFP), no Ca2+ increase was induced by H2O2 incubation (thin grey line). (C) Pooled data showing the increase in Ca2+ influx in TRPC1–caveolin-3-expressing cells upon Src activation (by H2O2). Graph represents mean ± s.e.m. of a minimum of 20 cells from four independent experiments. ***P<0.001.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Ca2+ influx through SACs is mediated by ROS in mdx fibres and Src inhibition reduces stretch-induced muscle damage in mdx mice. (A) Single fibres from mdx mice were loaded with the fluorescent Ca2+ indicator Fluo-4 to measure changes in [Ca2+]i. Fibres were then subjected to a series of ten stretched (eccentric) contractions without (control) or with the ROS scavenger Tiron (5 mM). (B) Changes in [Ca2+]i in single muscle fibres from mdx and WT mice were measured. Following the addition of 10 µM H2O2 for 30 minutes, fibres were co-incubated with H2O2 and the SAC blocker streptomycin (200 µM) for 40 minutes, and then both drugs were washed out. (C) EDL muscle was dissected from mdx mice and a series of ten stretched contractions was performed. Recovery of the muscle force was measured at three time points after stretched contractions (0, 30 and 60 minutes). Compared with control muscles incubated with PP3, inhibition of Src kinase by PP2 protected mdx muscle from damage induced by stretched contractions. (A) n=9 for control and n=6 for Tiron; (B) n=4 for WT and n=6 for mdx; (C) n=3 per group. *P<0.05; **P<0.01; ***P<0.001.

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