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Received December 14, 2006
Revised January 24, 2007
Accepted after revision February 15, 2007
1 St George's, University of London
* To whom correspondence should be addressed. E-mail: aalbert{at}sghms.ac.uk.
In the present work we used patch pipette techniques to study the properties of single Ca2+-permeable cation channels activated by the potent coronary vasoconstrictor ET- 1 in freshly dispersed rabbit coronary artery myocytes. With cell-attached recording bath application of 10 nM ET- 1 evoked cation channel currents (Icat) with sub- conductance states of about 18, 34 and 51 and 68 pS and a reversal potential of 0 mV. ET- 1 evoked channel activity when extracellular Ca2+ was the charge carrier illustrating significant Ca2+ permeability. ET- 1- induced responses were inhibited by ETA receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1-oleoyl- 2- acetyl- sn- glycerol (OAG) also stimulated Icat but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG- or ET- 1- induced Icat. Inositol 1, 4, 5- trisphosphate (IP3) did not activate Icat but greatly potentiated the response to OAG and this effect was blocked by heparin. Bath application of anti- TRPC3 and anti- TRPC7 antibodies to inside-out patches markedly inhibited ET- 1 evoked Icat but antibodies to TRPC1, C4, C5, and C6 had no effect. Immunocytochemical studies demonstrated preferential TRPC7 expression in the plasmalemma whereas TRPC3 was distributed throughout the myocyte and moreover co-localisation of TRPC3 and TRPC7 signals was observed at, or close to, the plasma membrane. Flufenamic acid, Gd3+, La3+ and extracellular Ca2+ inhibited Icat with IC50 values of 2.45 µM, 3.8 µM, 7.36 µM and 22 µM respectively. These results suggest that in rabbit coronary artery myocytes ET- 1 evokes a Ca2+-permeable non-selective cation channel with properties similar to TRPC3 and TRPC7 and indicates that these proteins may be important components of this conductance.
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