Related Experiment Video
Updated: Sep 30, 2026

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Published on: February 20, 2018
Characterization of G proteins involved in activation of nonselective cation channels by endothelin(B) receptor
Yoshifumi Kawanabe1, Nobuo Hashimoto, Tomoh Masaki
1Department of Neurosurgery, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8507, Japan. ykawanabe@rics.bwh.harvard.edu
Abstract:
1: 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)RDelta403). 2: 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)RDelta403 couple with G(13) and G(q), respectively. 3: 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)RDelta403. 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. 4: 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. 5: 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.
Related Concept Videos
Activation and Inactivation of G Proteins
G-Protein Gated Ion Channels
Sensory organs,...
GPCRs Regulate Adenylyl Cylase Activity
Two...
Transducer Mechanism: G Protein–Coupled Receptors
GPCRs are also called heptahelical, 7TM, or...
G Protein-coupled Receptors
G Protein-coupled Receptors
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...

