PMID:10215604
| Citation |
Hu, LA and King, SC (1999) 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. Biochem. J. 339 ( Pt 3):649-55 |
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| Abstract |
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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| Keywords |
4-Chloromercuribenzenesulfonate/metabolism; 4-Chloromercuribenzenesulfonate/pharmacology; Amines/metabolism; Amino Acid Substitution; Biological Transport/drug effects; Cell Membrane Permeability; Choline/metabolism; Consensus Sequence/genetics; Cysteine/genetics; Cysteine/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; Ethylmaleimide/metabolism; Ethylmaleimide/pharmacology; GABA Plasma Membrane Transport Proteins; Kinetics; Membrane Proteins/antagonists & inhibitors; Membrane Proteins/chemistry; Membrane Proteins/genetics; Membrane Proteins/metabolism; Membrane Transport Modulators; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Mesylates/metabolism; Mesylates/pharmacology; Models, Biological; Organic Anion Transporters; Polyamines/metabolism; Protein Conformation; Substrate Specificity; Sulfhydryl Compounds/metabolism; Sulfhydryl Reagents/chemistry; Sulfhydryl Reagents/metabolism; Sulfhydryl Reagents/pharmacology; gamma-Aminobutyric Acid/metabolism |
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