Cysteine-scanning Mutagenesis Reveals a Highly Amphipathic, Pore-lining Membrane-spanning Helix in the Glutamate Transporter GltT

Abstract

The carboxyl-terminal membrane-spanning segment 8 of the glutamate transporter GltT of Bacillus stearothermophilus was studied by cysteine-scanning mutagenesis. 21 single cysteine mutants were constructed in a stretch ranging from Gly-374 to Gln-404. Two mutants were not expressed, four were inactive, and two showed severely reduced glutamate transport activity. Cysteine mutations at the other positions were well tolerated. Only the two most amino-and carboxyl-terminal mutants (G374C, I375C, S399C, and Q404C) could be labeled with the large thiol reagent fluorescein maleimide, indicating unrestricted access and a location in a loop structure outside the membrane. The labeling pattern of these mutants using membrane-permeable and -impermeable thiol reagents showed that the N and C termini of the mutated stretch are located extra-and intracellularly, respectively. Thus, the location of the membranespanning segment was confined to a stretch of 23 residues between Gly-374 and Ser-399. Cysteine residues in three mutants in the central part of the segment (M381C, V388C, and N391C) could be labeled with the small and flexible reagent 2-aminoethyl methanethiosulfonate hydrobromide only, suggesting accessibility via a narrow aqueous pore. When the region was modeled as an ␣-helix, all positions at which cysteine mutations lead to inactive or severely impaired transporters cluster on one face of this helix. The inactive mutants showed neither proton motive force-driven uptake activity nor exchange activity nor glutamate binding. The results indicate that transmembrane segment 8 forms an amphipathic ␣-helix. The hydrophilic face of the helix lines an aqueous pore and contains many residues that are important for activity.Glutamate transporters in the mammalian central nervous system remove the neurotransmitter glutamate from the synaptic cleft into surrounding neurons and glial cells. Removal of glutamate prevents neurotoxicity of high concentrations of glutamate and helps to end the excitatory signal at some synapses (1-4). The proteins are secondary transporters that couple glutamate transport against the concentration gradient to the transport of protons, sodium ions, and potassium ions across the membrane. The glutamate transporters belong to a large family of transport proteins in which are also found bacterial glutamate transporters including GltT of Bacillus stearothermophilus (5). Computational analyses of the amino acid sequences and hydropathy profiles of the glutamate transporters showed that the proteins form a unique structural class of membrane proteins, which is structurally not related to any other family of secondary transporters (5, 6). Subsequent experimental studies confirmed that the proteins contain unique structural features, like water-filled pores and pore loops, which are not found in "regular" secondary transporters (7-11).The amino-terminal half of the transporters contains six membrane-spanning ␣-helices, with the amino terminus of the proteins located in the cytoplasm. The membrane topology of...

Arginine 445 Controls the Coupling between Glutamate and Cations in the Neuronal Transporter EAAC-1

“…The R445S phenotype is described equally well by either possibility. However, we favor the possibility in which the outer gate closure is affected, because if transmembrane domain 8 has a linear sequence in space (9,34), position 445 will be more extracellular than position 447.…”

Coupled, but Not Uncoupled, Fluxes in a Neuronal Glutamate Transporter Can Be Activated by Lithium Ions

“…Possible candidates are the essential asparagine 366 and aspartate 368 (Fig. 1) as well as essential aspartate residues in transmembrane domain 8 (28,40). Asparagine 366 and aspartate 368 would be close, at least in the primary structure, to threonine 370.…”

Structural model for γ-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site

“…After 3 min, the membranes were recovered by centrifugation (20,000 ϫ g for 15 min at 4°C), resuspended in PBS, mixed with sample buffer with or without 10 mM DTT, and subjected to SDS͞PAGE (10% acrylamide) and Western blot analysis. For sulfhydryl modification, two methanethiosulfonate reagents were used, 2-(trimethylammonium)ethyl methanethiosulfonate bromide and sodium (2-sulfonatoethyl)methanethiosulfonate, under described conditions (18,43) (8) and the recombinant expressed receptor (100 g protein) in PBS (500 l final volume) (6). After incubation for 90 min at 25°C, the samples were filtered through GF͞B filters (presoaked in 0.2% polyethyleneimine for 3 h) and rinsed three times with ice-cold saline (0.9% NaCl).…”