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4-Aminobutyrate (GABA) transporters from the amine-polyamine-choline superfamily: substrate specificity and ligand recognition profile of the 4-aminobutyrate permease from Bacillus subtilis.

ABSTRACT: A previous study [Ferson, Wray and Fisher (1996) Mol. Microbiol. 22, 693-701] has shown that transposon-mediated disruption of a protein 47% identical to the Escherichia coli GABA (4-aminobutyrate) transporter abolishes the ability of nitrogen-limited culture conditions to induce expression of a GABA transport activity in Bacillus subtilis. Here it is demonstrated directly that the B. subtilis GABA permease (gabP) gene can complement the transport defect in the gabP-negative E. coli strain. Unexpectedly, the ligand-recognition profile of the B. subtilis GabP was found to differ substantially from that of the highly homologous E. coli GabP. Unlike the E. coli GabP, the B. subtilis GabP: (i) exhibits approx. equal preference for the 3-carbon (beta-alanine, Km=9.6 microM) and the 4-carbon (GABA, Km=37 microM) amino acids, and (ii) resists inhibition by bulky, conformationally constrained compounds (e.g. nipecotic acid, guvacine), which are active against GABA transporters from brain. The present study shows additionally that the B. subtilis GabP can translocate several open-chain GABA analogues (3-aminobutyrate, 3-aminopropanoate, cis-4-aminobutenoate) across the membrane via counterflow against [3H]GABA. Thus, consistent with the idea that the ligand-recognition domain of the B. subtilis GabP is less spacious than that of the close homologue from E. coli, the former exhibits more stringent requirements than the latter for substrate recognition and translocation. These distinct functional characteristics of the E. coli and B. subtilis GABA transporters provide a basis by which to identify ligand-recognition domains within the amine-polyamine-choline transporter superfamily.

SUBMITTER: Brechtel CE

PROVIDER: S-EPMC1219618 | biostudies-other | 1998 Aug

REPOSITORIES: biostudies-other

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Project description:The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].

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4-Aminobutyrate (GABA) transporters from the amine-polyamine-choline superfamily: substrate specificity and ligand recognition profile of the 4-aminobutyrate permease from Bacillus subtilis.

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