Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C45
Functional splicing of TRPC channels in human aortic endothelial cells
B. Zeng1, S. Atkin1, S. Xu1
1. Hull York Medical School, University of Hull, Hull, United Kingdom.
The transient receptor potential (TRP) channels are Ca2+-permeable cationic channels. The subfamily of TRPC has been suggested as the molecular basis of receptor-operated channels and/or store-operated channels. Ca2+ influx through TRPC channels has been demonstrated to play a critical role in the physiology and pathophysiology of endothelial cells (Freichel et al., 2001; Watanabea et al., 2008). The alternative splicing activity of TRPC channels are high, especially for TRPC4 channel. So far eight TRPC4 isoforms have been reported, in which six have six transmembrane segments, and the other two are truncated. TRPC4 shares similarities to TRPC5 in sequence and functional properties (Xu et al., 2008), and they both form heteromultimers with TRPC1, which has more than three isoforms reported in human. The functional mechanisms of TRPC channels and their isoforms in endothelial cells are unclear. In this study, we are intended to examine the expression profile of TRPC channels and their functionality in human aortic endothelial cells (HAECs). HAECs were purchased from PromoCell (Germany) and primarily cultured in the endothelial cell growth medium. RT-PCR with primer sets specific for different TRPC genes and potential splicing sites were used to detect the existence of TRPCs and their splicing. All PCR products were sequenced to confirm the presence or absence of certain exons. The functionality of TRPC channels on the interleukin-6 (IL-6) secretion and proliferation in HAECs was investigated using IL-6 ELISA and WST-1 assay, respectively. TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 channels were expressed in HAECs, but TRPC7 was undetectable. Three alternatively spliced isoforms for TRPC1, three isoforms for TRPC4 and two isoforms for TRPC6 were detected and confirmed by sequencing. Overexpression of TRPC1 and TRPC4 in HAECs increased cell proliferation. Application of blocking antibodies targeting to TRPC1 and TRPC4 significantly inhibited the cell proliferation. The secretion of IL-6 was significantly reduced by overexpressing TRPC1 or TRPC4 in HAECs. The spliced isoform of TRPC1 altered the secretory function if cotransfected with TRPC4. Our results provide the evidence of TRPC channels in the secretory function and proliferation in HAECs, suggesting their important roles in regulating endothelial function which may relate to the inflammatory reaction and pathogenesis of cardiovascular diseases.
Where applicable, experiments conform with Society ethical requirements