Enhancing Glutamate Transport: Mechanism of Action of Parawixin1, a Neuroprotective Compound from Parawixia bistriata Spider Venom

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 potassiumbound 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.

Astroglia as a cellular target for neuroprotection and treatment of neuro‐psychiatric disorders

“…Parawixin1, purified from the venom of the spider parawixia bistriata , enhances directly and selectively EAAT2 function by facilitating conformational transitions involved in substrate translocation (Fontana et al, 2007). Site‐directed mutagenesis identified a structural region within EAAT2 which is important for the transporter‐enhancing activity in transmembrane domains 2, 5, and 8 (Mortensen, Liberato, Coutinho‐Netto, Dos Santos, & Fontana, 2015).…”

Amino Acid Transporters in Drug Discovery

“…The expression profile of this protein is quite broad: kidney, intestine, brain, lung, skeletal muscle, placenta and pancreas (Broer and Brookes, 2001;Deitmer et al, 2003;Gliddon et al, 2009;Kekuda et al, 1996;Utsunomiya-Tate et al, 1996). ASCT2 function has been extensively studied using different experimental approaches: cell systems (Torres-Zamorano et al, 1998) as well as proteoliposome reconstituted with the rat transporter extracted from kidney brush borders (Oppedisano et al, 2004;2007) and, more recently, with the human isoform over-expressed in P.pastoris . ASCT2 is an amino acid transporter whose acronym stands for AlaSerCysTransporter 2, even though, it is now well recognized that it has a primary role as glutamine transporter.…”

Glutamate Transporter Activators as Anti-Nociceptive Agents

“…Given the marked therapeutic potential of enhancement of glutamate transporters, there are ongoing efforts of drug screening for potent and selective regulators of glutamate levels [30][31][32].…”