TRPCs are calcium-permeable cation channels that respond to calcium-store depletion or receptor-dependent activation of phospholipase C (Montell et al. 2002). We have shown that TRPC1 is expressed in a range of blood vessels and is a subunit of store-operated channels in smooth muscle cells of cerebral arterioles (Xu & Beech, 2001). We are now exploring if TRPC1 is associated with other TRPCs in blood vessels. In this study we focused on TRPC5. Male 6- to 8-week-old mice were killed by cervical dislocation according to Schedule 1 procedures. Discarded pieces of human blood vessel were obtained anonymously and with ethical approval from coronary artery bypass operations.

Western blotting was performed on lysates from mouse aorta and the medial layer of human saphenous vein (SV) (n = 3). Anti-TRPC1 polyclonal antibody labelled a protein of 90 kDa, close to the predicted mass of 87-93 kDa. Anti-TRPC5 antibodies generated in rabbit or chicken labelled a protein of 140 kDa. In mouse brain lysates, anti-TRPC5 antibody similarly labelled a protein of 140 kDa. After deglycosylation with peptide N-glycosidase F (n = 3) the protein reduced in size to 110 kDa, very close to the predicted mass of 110-111 kDa. In mouse and human TRPC5 sequences a consensus N-linked glycosylation site is predicted between the third and fourth membrane-spanning segments. Deglycosylation did not have an effect on TRPC1, which lacks a strong consensus N-linked glycosylation site. Vascular smooth muscle cells in sections of human SV and radial artery were stained by anti-TRPC1 and anti-TRPC5 antibodies, with no staining occurring in the presence of pre-immune serum or in the absence of primary antibody. Co-immunoprecipitation studies were performed in the presence of 1 % Triton X-100 on lysates from tsA cells transiently co-expressing FLAG epitope tagged-TRPC1 and TRPC5. Anti-FLAG antibody precipitated TRPC1 and TRPC5, as did anti-TRPC5 antibody (n = 2), confirming the results of Strubing et al. (2001). Using polyclonal antibody to TRPC1, similar results were found in lysates of human SV and mouse brain (n = 2 for each).

The data suggest that TRPC5 is a glycosylated protein and that it is expressed along with TRPC1 in vascular smooth muscle. We confirm that TRPC1 and TRPC5 co-immunoprecipitate after heterologous expression, and also show their co-association in the medial layer of human vein.

We thank The Wellcome Trust for support and are grateful to D. Clapham and G. Barritt for rabbit anti-TRPC5 and anti-TRPC1 antibodies, and C. Montell and Y. Mori for TRPC1 and TRPC5 cDNAs.

All procedures accord with current UK legislation.