Mammalian counterparts of the Drosophila trp genes are suggested to encode non-selective and Ca2+-permeable channels such as store- and receptor-operated channels (Birnbaumer et al. 2000). We have shown that mammalian TrpC1 is a plasma membrane protein in vascular smooth muscle cells and a functional subunit of store-operated Ca2+ channels (Xu & Beech, 2001). However, a TrpC1 blocking antibody produced only partial inhibition of store-operated Ca2+ entry, suggesting involvement of other TrpC proteins. In this study we investigated the expression of seven additional trp genes at mRNA and, in some cases, protein levels. We used solution RT-PCR, in situ RT-PCR and western blotting applied to fresh vascular tissue or brain. Trp1, 2, 4 and 7 PCR products were identified by sequencing, others by molecular size only. No products were detected in RT-PCR experiments if reverse transcriptase (RT) reaction was omitted. TrpC3 and TrpC6 antibodies were from Alomone Labs. Each result is representative of ▓ge│ 3 independent observations. Mice were killed by gradually increasing inhaled CO2 to excess, and rabbits killed by I.V. injection of 70 mg kg-1 pentobarbitone. Discarded human tissue was obtained anonymously with ethical permission from the Yorkshire Heart Centre.
Messenger RNAs for trp1-7 and LTRP 2 were detected in mouse brain and rabbit pial membrane. Trp1, trp 3-6 and LTRP 2 were also positive in arterioles isolated from pial membrane, and trp1, trp 4, trp 6 and trp 7 were positive in human radial artery. Trp 1 and LTRP 2 mRNAs were detected in single pial arteriolar smooth muscle cells harvested by a micro-pipetting method. Localisation of trp 1 mRNA to smooth muscle cells was confirmed by in situ RT-PCR applied to paraffin-embedded sections of human left internal mammary artery. Proteins of the expected size were labelled by TrpC antibodies: TrpC1 (Xu & Beech, 2001), TrpC3 (mouse brain, pial membrane, portal vein and aorta), TrpC6 (mouse brain and aorta, and human aorta) and TrpC7 (human aorta).
The data indicate that trp genes in addition to trp1 are expressed as mRNA and protein in a range of vascular tissues. Thus heteromultimers of these trp gene products with TrpC1 may occur.
S.Z.X. has an ORS award and research funds were from the National Heart Research Fund and Wellcome Trust.
- Birnbaumer, L., Boulay, G., Brown, D., Jiang, M., Dietrich, A., Mikoshiba, K., Zhu, X. & Qin, N. (2000). Recent Prog. Hormone Res. 55, 127-161.
Xu, S.Z. & Beech, D.J. (2001). Circ. Res. 88, 84-87.