title: Conformational changes monitored on the glutamate transporter GLT-1 indicate the existence of two neurotransmitter-bound states.
abstract: Membrane vesicles from rat brain have been subjected to trypsin treatment in the absence and presence of substrates of the (Na+ + K+)-coupled L-glutamate transporter GLT-1. The fragments of this transporter have been detected upon immunoblotting employing several antibodies raised against sequences from this transporter. At the amino terminus, initially a fragment of an apparent molecular mass of 30 kDa is generated. This fragment is subsequently cleaved to one of 16 kDa. The generation of these bands is greatly inhibited in the presence of lithium. Moreover, lithium abolishes the positive cooperative activation of the transporter by sodium. The generation of the 30- and 16-kDa fragments is accelerated in the presence of L-glutamate and other transportable analogues, provided sodium is present as well. The 30-kDa fragment also contains an epitope from the loop connecting the putative membrane-spanning alpha-helices 3 and 4. This epitope, in contrast with the amino-terminal one, is destroyed with time. The carboxyl-terminal epitope is predominantly located on a 43-kDa fragment which is slowly converted to one of 35 kDa. This conversion is not inhibited by lithium. It is, however, stimulated by L-glutamate and other transportable analogues, but only in sodium-containing media. Potassium also stimulates this conversion regardless of the presence of L-glutamate. The stimulation of generation of amino- and carboxyl-terminal fragments by L-glutamate is not mimicked by the nontransportable analogue dihydrokainate. However, the analogue blocks the stimulation exerted by L-glutamate. In addition to new experimental information on the transporters topology, our observations provide novel information on the function of the GLT-1 transporter. Although lithium by itself does not sustain transport, it may occupy one of the sodium sites and be transported. Furthermore, the transporter-glutamate complex appears to exist in at least two states. After the initial binding (suggested to be important for the decay of synaptic glutamate), it undergoes a conformational change which represents, or is tightly associated with, the transport step.
authors.affiliation: Department of Biochemistry, Hadassah Medical School, Hebrew University, Jerusalem, Israel.
authors.forename: M
authors.initials: M
authors.lastname: Grunewald
authors.forename: B
authors.initials: B
authors.lastname: Kanner
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D018698:Glutamic Acid / Q000378:metabolism*; D007608:Kainic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008094:Lithium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats
chemical_list: D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D010446:Peptide Fragments; D018698:Glutamic Acid; C020889:dihydrokainic acid; D008094:Lithium; D007608:Kainic Acid