Department of Chemistry, Binghamton University, Binghamton, New York 13902.
Department of Chemistry, Binghamton University, Binghamton, New York 13902.
J Biol Chem. 2019 Aug 9;294(32):12180-12190. doi: 10.1074/jbc.RA119.009421. Epub 2019 Jun 24.
Plasma membrane-associated glutamate transporters play a key role in signaling by the major excitatory neurotransmitter glutamate. Uphill glutamate uptake into cells is energetically driven by coupling to co-transport of three Na ions. In exchange, one K ion is counter-transported. Currently accepted transport mechanisms assume that Na and K effects are exclusive, resulting from competition of these cations at the binding level. Here, we used electrophysiological analysis to test the effects of K and Na on neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1; the rat homologue of human excitatory amino acid transporter 3 (EAAT3)). Unexpectedly, extracellular K application to EAAC1 induced anion current, but only in the presence of Na This result could be explained with a K/Na co-binding state in which the two cations simultaneously bind to the transporter. We obtained further evidence for this co-binding state, and its anion conductance, by analyzing transient currents when Na was exchanged for K and effects of the [K]/[Na] ratio on glutamate affinity. Interestingly, we observed the K/Na co-binding state not only in EAAC1 but also in the subtypes EAAT1 and -2, which, unlike EAAC1, conducted anions in response to K only. We incorporated these experimental findings in a revised transport mechanism, including the K/Na co-binding state and the ability of K to activate anion current. Overall, these results suggest that differentiation between Na and K does not occur at the binding level but is conferred by coupling of cation binding to conformational changes. These findings have implications also for other exchangers.
质膜相关的谷氨酸转运体在作为主要兴奋性神经递质谷氨酸的信号转导中发挥着关键作用。细胞内的谷氨酸摄取是通过与三种钠离子共转运来驱动的，这是一种能量依赖性的过程。在交换过程中，一个钾离子被反向转运。目前公认的转运机制假设钠和钾的作用是相互排斥的，这是由于这些阳离子在结合水平上的竞争造成的。在这里，我们使用电生理分析来测试钾和钠对神经元谷氨酸转运体兴奋性氨基酸载体 1（EAAC1；人类兴奋性氨基酸转运体 3（EAAT3）的大鼠同源物）的影响。出乎意料的是，细胞外钾的应用诱导 EAAC1 产生阴离子电流，但只有在存在钠离子的情况下。这个结果可以用钾/钠共结合状态来解释，在这种状态下，两个阳离子同时与转运体结合。我们通过分析当钠离子被钾离子取代时的瞬态电流以及[K]/[Na]比值对谷氨酸亲和力的影响，获得了这种共结合状态及其阴离子电导的进一步证据。有趣的是，我们不仅在 EAAC1 中观察到了钾/钠共结合状态，也在亚型 EAAT1 和 -2 中观察到了这种状态，与 EAAC1 不同的是，这两种亚型仅对钾离子响应而产生阴离子电流。我们将这些实验发现纳入一个修订后的转运机制中，包括钾/钠共结合状态和钾激活阴离子电流的能力。总的来说，这些结果表明，在结合水平上并没有区分钠离子和钾离子，而是通过阳离子结合与构象变化的偶联来实现的。这些发现对其他交换器也有影响。
