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

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Abstract
We demonstrated recently that in Chinese hamster ovary cells stably expressing human recombinant endothelinA receptors (CHO-ETAR), 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-{β-[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-ETAR couples with G12 in addition to Gq and Gs. 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 ETARs with coupling to either Gqor Gs/G12 (designated ETARΔ385 and SerETAR, respectively) and a dominant-negative mutant of G12 (G12G228A). ETARΔ385 is truncated immediately downstream of Cys385 in the C terminus as palmitoylation sites, whereas SerETAR is unpalmitoylated because of substitution of all the cysteine residues to serine (Cys383Cys385–388 → Ser383Ser385–388). In CHO-ETARΔ385, stimulation with ET-1 activated only SOCC. In CHO-SerETAR or CHO-ETAR pretreated withU73122, 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-SerETAR. Microinjection of G12G228A abolished the activation of NSCC-1 and NSCC-2 in CHO-ETAR and that of NSCC-1 in CHO-SerETAR. These results indicate that ETAR activates three types of Ca2+ channels via different G protein-related pathways. NSCC-1 is activated via a G12-dependent pathway, NSCC-2 via Gq/PLC- and G12-dependent pathways, and SOCC via a Gq/PLC-dependent pathway.