Characterization of G proteins involved in activation of nonselective cation channels by endothelin B receptor

We recently demonstrated that endothelin‐1 (ET‐1) activates two types of Ca 2+ ‐permeable nonselective cation channels (NSCC‐1 and NSCC‐2) in Chinese hamster ovary cells expressing endothelin B receptors (CHO‐ET B R) that couple with G q and G i . The purpose of the present study was to identify the G proteins involved in the activation of these Ca 2+ channels by ET‐1. For this purpose, we constructed CHO cells expressing an unpalmitoylated (Cys 402 Cys 403 Cys 405 →Ser 402 Ser 403 Ser 405 ) ET B R (CHO‐SerET B R) and ET B R truncated at the cytoplasmic tail downstream of Cys 403 (CHO‐ET B RΔ403). Based on the data obtained from actin stress fibre formation, CHO‐ET B R couple with G 13 . Therefore, CHO‐ET B R couple with G q , G i and G 13 . CHO‐SerET B R and CHO‐ET B RΔ403 couple with G 13 and G q , respectively. ET‐1 activated NSCC‐1 in CHO‐ET B R preincubated with phospholipase C (PLC) inhibitor, U73122, and in CHO‐SerET B R. On the other hand, ET‐1 failed to activate Ca 2+ channels in CHO‐ET B RΔ403. Microinjection of dominant negative mutants of G 13 (G 13 G225A) abolished activation of NSCC‐1 and NSCC‐2 in CHO‐ET B R and that of NSCC‐1 in CHO‐SerET B R. Y‐27632, a specific Rho‐associated kinase (ROCK) inhibitor, did not affect the ET‐1‐induced transient and sustained increase in [Ca 2+ ] i in CHO‐ET B R. These results indicate that (1) the cytoplasmic tail downstream of the palmitoylation sites of ET B R, but not the palmitoylation site itself, is essential for coupling with G 13 , (2) the activation mechanism of each Ca 2+ channel by ET‐1 is different in CHO‐ET B R. NSCC‐1 activation depends on G 13 ‐dependent cascade, and NSCC‐2 activation depends on both G q /PLC‐ and G 13 ‐dependent cascades. Moreover, ROCK‐dependent cascade is not involved in the activation of these channels.British Journal of Pharmacology (2002) 136 , 1015–1022. doi: 10.1038/sj.bjp.0704805