Enhancing glutamate transport:: Mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom
Enhancing glutamate transport:: Mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom
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DOI：
10.1124/mol.107.037127
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发表时间：
2007-11-01
影响因子：
3.6
通讯作者：
Amara, Susan G.
中科院分区：
文献类型：
--
作者：
Cristina, Andreia;Fontana, Karklin;Amara, Susan G.
先前的研究表明，从蜘蛛 Parawixia bistriata 毒液中纯化的化合物可以刺激神经胶质谷氨酸转运蛋白的活性，并可以保护视网膜组织免受缺血性损伤。为了了解该化合物增强转运的机制，我们检查了其在脂质体和转染的 COS-7 细胞中溶解和重建后对谷氨酸转运蛋白功能特性的影响。在这里，我们在这两个系统中证明，Parawixin1 通过不改变共底物谷氨酸或钠的表观亲和力的机制，促进 EAAT2 转运蛋白亚型直接和选择性增强谷氨酸流入。在脂质体中，当细胞外钠和细胞内钾浓度处于生理范围内时，我们观察到 Parawixin1 的最大增强作用。此外，当细胞外钾升高且钠梯度降低时，该化合物不会在有利于流出的离子条件下增强谷氨酸的逆向转运，也不会在内部钾不存在的情况下改变谷氨酸的交换。这些观察结果表明，Parawixin1 促进钾结合转运蛋白的重新定向，这是运输循环中的限速步骤，实验进一步支持了这一结论，表明 Parawixin1 不会刺激钾依赖性重新定向步骤中有缺陷的 EAAT2 转运突变体 (E405D) 的摄取。因此，Parawixin1 通过一种新机制增强转运，该机制针对转运循环中不同于底物流入或流出的步骤，并为设计变构作用于转运蛋白以增加谷氨酸清除率的新药物提供了基础。
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.