Vol. 62, Issue 1, 75-80, July 2002

Molecular Mechanisms for the Activation of Voltage-Independent Ca²⁺ Channels by Endothelin-1 in Chinese Hamster Ovary Cells Stably Expressing Human Endothelin_A Receptors

Yoshifumi Kawanabe, Yasuo Okamoto, Soichi Miwa, Nobuo Hashimoto, and Tomoh Masaki

Departments of Neurosurgery (Y.K., N.H.) and Pharmacology (Y.K., Y.O., S.M., T.M.), Kyoto University Faculty of Medicine, Kyoto, Japan

We demonstrated recently that in Chinese hamster ovary cells stably expressing human recombinant endothelin_A receptors (CHO-ET_AR), endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca²⁺ channel (SOCC), which can be distinguished by Ca²⁺ channel blockers such as 1-{-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl}-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate (LOE 908). We also reported that CHO-ET_AR couples with G₁₂ in addition to G_q and G_s. The purpose of the present study was to identify the G proteins involved in the activation of these Ca²⁺ channels by ET-1, using mutated ET_ARs with coupling to either G_q or G_s/G₁₂ (designated ET_AR385 and SerET_AR, respectively) and a dominant-negative mutant of G₁₂ (G₁₂G228A). ET_AR385 is truncated immediately downstream of Cys³⁸⁵ in the C terminus as palmitoylation sites, whereas SerET_AR is unpalmitoylated because of substitution of all the cysteine residues to serine (Cys³⁸³Cys^385-388  Ser³⁸³Ser^385-388). In CHO-ET_AR385, stimulation with ET-1 activated only SOCC. In CHO-SerET_AR or CHO-ET_AR pretreated with U73122, an inhibitor of phospholipase C (PLC), ET-1 activated only NSCC-1. Dibutyryl cAMP alone did not activate any Ca²⁺ channels in the resting and ET-1-stimulated CHO-SerET_AR. Microinjection of G₁₂G228A abolished the activation of NSCC-1 and NSCC-2 in CHO-ET_AR and that of NSCC-1 in CHO-SerET_AR. These results indicate that ET_AR activates three types of Ca²⁺ channels via different G protein-related pathways. NSCC-1 is activated via a G₁₂-dependent pathway, NSCC-2 via G_q/PLC- and G₁₂-dependent pathways, and SOCC via a G_q/PLC-dependent pathway.