ACTIVATION OF THE CALCIUM-PERMEABLE CATION CHANNEL CD20 BY ALPHA-SUBUNITS OF THE G(I) PROTEIN

When the calcium-permeable cation channel CD20 is expressed in Balb/c 3T3 cells, it is activated by insulin-like growth factor-I (IGF-I) via the IGF-I receptor (Kanzaki, M., Nie, L., Shibata, H., and Kojima, I. (1997) J. Biol. Chem. 272, 4964-4969). The present study was conducte d to investigate the role of G proteins in the regulation of the CD20 channel. In the excised patch clamp mode, activation of the CD20 chann el by IGF-I required GTP, Mg2+, and ATP in the bath solution, and remo val of either GTP or ATP attenuated the activation. Non-hydrolyzable A TP could substitute for ATP, and guanyl-5'-yl thiophosphate blocked th e activation of the channel by IGF-I. The CD20 channel was also activa ted by guanosine 5'-3-O-(thio)triphosphate, and ATP was not required f or the activation. Addition of a preparation of G(i)/G(o) holoprotein purified from bovine brain activated the CD20, and the beta-adrenergic receptor kinase peptide did not affect the number of channel openings induced by the G protein. The CD20 channel was stimulated by the GTP- bound form of recombinant G(i2) alpha subunit purified from Sf9 cells. The G(i1) alpha subunit was less effective, and the G(i1) alpha subun it had no effect. Purified recombinant beta(1) gamma(2) subunits did n ot affect the activity of the channel. Finally, IGF-I-induced activati on of CD20 was inhibited by an antibody against G(i2) alpha subunit. T hese findings indicate that the CD20 channel expressed in Balb/c 3T3 c ells is activated by the IGF-I receptor via the alpha subunits of hete rotrimeric G proteins.