Abstract
Previous studies have shown that a compound purified from the spider Parawixia bistriata venom stimulates the activity of glial glutamate transporters and can protect retinal tissue from ischemic damage. To understand the mechanism by which this compound enhances transport, we examined its effects on the functional properties of glutamate transporters after solubilization and reconstitution in liposomes and in transfected COS-7 cells. Here, we demonstrate in both systems that Parawixin1 promotes a direct and selective enhancement of glutamate influx by the EAAT2 transporter subtype through a mechanism that does not alter the apparent affinities for the cosubstrates glutamate or sodium. In liposomes, we observed maximal enhancement by Parawixin1 when extracellular sodium and intracellular potassium concentrations are within physiological ranges. Moreover, the compound does not enhance the reverse transport of glutamate under ionic conditions that favor efflux, when extracellular potassium is elevated and the sodium gradient is reduced, nor does it alter the exchange of glutamate in the absence of internal potassium. These observations suggest that Parawixin1 facilitates the reorientation of the potassium-bound transporter, the rate-limiting step in the transport cycle, a conclusion further supported by experiments showing that Parawixin1 does not stimulate uptake by an EAAT2 transport mutant (E405D) defective in the potassium-dependent reorientation step. Thus, Parawixin1 enhances transport through a novel mechanism targeting a step in the transport cycle distinct from substrate influx or efflux and provides a basis for the design of new drugs that act allosterically on transporters to increase glutamate clearance.
Footnotes
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This work has been supported by National Institutes of Health grants NS33272 and MH080726 (to S.G.A.). N.C.D. acknowledges support from the Norwegian Top Research Program (Toppforskningsprogrammet) and the Norwegian Research Council.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.037127.
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ABBREVIATIONS: CNS, central nervous system; EAAT, human excitatory amino acid transporter; GLT-1, rat glutamate transporter 1; MS-153, (R)-(–)-5-methyl-1-nicotinoyl-2-pyrazoline; GPI-1046, 3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate; FK506, tacrolimus; EAAT2, human glutamate transporter 2, homologous to GLT-1; D-PBS, Dulbecco's phosphate-buffered saline; U, unit of toxin based on λ = 215nm (1 OD = 1000 U); NaSCN, sodium thiocyanate; ANOVA, analysis of variance; DHK, dihydrokainate.
- Received April 19, 2007.
- Accepted July 23, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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