C-terminal interactions modulate the affinity of GLAST glutamate transporters in salamander retinal glial cells.
1. Proteins that interact with the intracellular carboxy termini of neurotransmitter- and voltage-gated ion channels are known to control the subcellular localization of the channels, localize other proteins near those channels, and modulate channel activity. By contrast, little is known about the control of neurotransmitter transporter function by interacting proteins. 2. To competitively disrupt interactions of the C- and N-termini of the GLAST glutamate transporter with other proteins, we dialysed whole-cell patch-clamped retinal glia with peptides identical to the eight amino acids at the C- or N-termini of the transporter, and compared the effect on transporter-mediated currents with dialysis of scrambled versions of the same peptides. 3. Dialysis with the N-terminus peptide had no effect on the maximum glutamate-evoked current nor on the glutamate affinity of the transporter. Dialysis with the C-terminus peptide had no effect on the maximum current, but increased the affinity of the transporter for glutamate (compared with scrambled C-terminus peptide, and with N- and scrambled N-terminus peptides: Km decreased from 16 to 11 microM)). 4. These data suggest that disruption of an interaction between an intracellular protein and the last eight amino acids of the GLAST C-terminus, which have some similarity to the PDZ binding domain of ion channel C-termini, increases the glutamate affinity of GLAST. Thus, the interacting protein decreases the affinity of GLAST transporters. 5. Removing the GLAST C-terminus interaction increases the transporter current by 40 % at low glutamate concentrations. Thus, this interaction may significantly slow the removal of low concentrations of glutamate from the extracellular space, and affect the kinetics of retinal cell light responses.
Marie
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Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK.
Attwell
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D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001667:Binding, Competitive; D004594:Electrophysiology; D018698:Glutamic Acid / Q000494:pharmacology; D007700:Kinetics; D009457:Neuroglia / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding; D012160:Retina / Q000378:metabolism*; D014562:Caudata
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D010446:Peptide Fragments; D018698:Glutamic Acid