Recombinant αi-3 Subunit of G Protein Activates Gk-gated K+ Channels

R Mattera,A Yatani,G E Kirsch,R Graf,K Okabe,J Olate,J Codina,A M Brown,L Birnbaumer

doi:10.1016/s0021-9258(17)31281-4

R Mattera, A Yatani + Show 7 more

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https://doi.org/10.1016/s0021-9258(17)31281-4

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Abstract

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.

Highlights

G proteins, those sensitive to pertussis express from one to four types of Ga, molecules of 379-395 toxin, a r e difficult to separatebiochemically, creating amino acids, which are substrates for cholera toxin,from one uncertainty in functional assignments. For this reasonto three typesof Gai molecules called Gai-1, Gai-2, and Gaithe cDNAs encoding Gai-3 and two of the Gas splice 3, which are 354 or 355 amino acids in length, and substrates variants were expressed as fusion proteins in Esche- for pertussis toxin(PTX) and one tworo types of a0 molecules richia coli using a T 7 promoter-based expression sys- ( P T X substrates) which are 354 amino acids in length tem
PlasrnidlPhage Method-E. coli cells containing pT7-a. plasmids were thawed, grown at 37 "C in M9 medium supplemented with 0.2 mMof each amino acid and 25 pg/ml ampicillin
In the present studieswe modified as needed the GacDNA moleculesby site-directed mutagenesis to retaina unique C’CATGG NcoI restriction siteat theinitiation of their open reading frames and inserted them intothe BumHI site of the cloning cassette of the pT7-7 expression plasmid developed by Tabor andRichardson [16,17,18].Part of the nucleotide composition of the 5’ region that flanks the Ga! ORFs is as follows, of pT7-7

Summary

Introduction

Determination of Atrial G Protein-gated K + Channel Activity-Single atrial K+ channel currents in excised inside-out membrane patches from adult guinea pigs, neonatal rat,and cultured chick embryos were recorded by the patch clamp method of Hamill et al [24] using recording conditions and equipment described previously [5,6,7,8,9]. Rai-3 presentin lysate supernatants was tested for its activity on Gk-gated K' channels in inside-out atrial membrane patches from adult guinea pig, 1-3 day neonatal rat and 1214-day-old chick embryos [9] and found to stimulate single K+ channel currents in these systems.

Results

Conclusion