Recombinant alpha i-3 subunit of G protein activates Gk-gated K+ channels.

G proteins, particularly those sensitive to pertussis toxin, are difficult to separate biochemically, creating uncertainty in functional assignments. For this reason the cDNAs encoding G alpha i-3 and two of the G alpha s splice variants were expressed as fusion proteins in Escherichia coli using a T7 promoter-based expression system. These proteins were denoted r alpha i-3 and r alpha s (short and long) and accumulated in bacteria to as much as 5-10% of total cellular protein, of which 5-10% was soluble in lysates. Soluble r alpha subunits were tested for stimulation of K+ channel activity in inside-out atrial membrane patches and for reconstitution of cyc- adenylyl cyclase activity. r alpha i-3, activated either by guanosine 5'-(3-thio)triphosphate (GTP gamma S) or AlF-4, stimulated in a concentration-dependent manner single channel K+ currents in isolated atrial membrane patches of three species: guinea pigs, neonatal rats, and embryonic chick. In contrast, GTP gamma S-activated r alpha s did not. In agreement with a similar study by Graziano et al. (Graziano, M. P., Casey, P. J. and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375-11381), both r alpha s forms reconstituted GTP gamma S-stimulated cyc- adenylyl cyclase activity, albeit at concentrations 50-100 times higher than those needed with native Gs. The concentrations of r alpha i-3 needed to stimulate the K+ channels were also higher than needed with native human erythrocyte Gk, in this case 30-50 times. Single K+ channel currents stimulated by r alpha i-3 were indistinguishable from those stimulated by the natural effector acetylcholine. Thus, bacterial expression of G alpha subunits provided the means to demonstrate unequivocally that Gi-3 has intrinsic Gk activity.