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.
Mattera
R
R
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Yatani
A
A
Kirsch
G E
GE
Graf
R
R
Okabe
K
K
Olate
J
J
Codina
J
J
Brown
A M
AM
Birnbaumer
L
L
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016275:Atrial Function; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D006168:Guinea Pigs; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D015221:Potassium Channels / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology*
D004251:DNA Transposable Elements; D046911:Macromolecular Substances; D015221:Potassium Channels; D011994:Recombinant Proteins; D004247:DNA; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol