Identification of the amine-polyamine-choline transporter superfamily 'consensus amphipathic region' as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents. Role of Cys-300 in restoring thiol sensitivity to Gabp lacking Cys.
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].
Hu
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Department of Physiology and Biophysics, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0641, USA.
King
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D002731:4-Chloromercuribenzenesulfonate / Q000378:metabolism / Q000494:pharmacology; D000588:Amines / Q000378:metabolism; D019943:Amino Acid Substitution; D001692:Biological Transport / Q000187:drug effects; D002463:Cell Membrane Permeability; D002794:Choline / Q000378:metabolism; D016384:Consensus Sequence / Q000235:genetics*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008698:Mesylates / Q000378:metabolism / Q000494:pharmacology; D008954:Models, Biological; D027361:Organic Anion Transporters; D011073:Polyamines / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000588:Amines; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D008698:Mesylates; D027361:Organic Anion Transporters; D011073:Polyamines; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C513601:gabP protein, E coli; C074502:methanethiosulfonate; D005680:gamma-Aminobutyric Acid; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine; D002794:Choline; D005033:Ethylmaleimide