Abstract
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.
Highlights
Plasma membrane–associated glutamate transporters play a key role in signaling by the major excitatory neurotransmitter glutamate
Thought to be transported in the negatively charged form [3,4,5], this stoichiometry predicts that two positive charges move into the cell with each glutamate molecule, giving rise to a coupled transport current [5, 6]. Consistent with this prediction, transport current induced by glutamate application to glutamate transporter (excitatory amino acid transporter (EAAT))– expressing cells has been observed in many reports [7,8,9,10]
The simulation parameters for relative anion currents listed in the legend of Fig. 8B. These results suggest that the Kϩ/Naϩ co-binding state is present in all three transporter subtypes, but its effect on overall anion current is masked by the large conductance of the Kϩ-bound state in EAAT1 and -2
Summary
A K؉/Na؉ co-binding state: Simultaneous versus competitive binding of K؉ and Na؉ to glutamate transporters. 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. We measured anion current in the presence of varying concentrations of extracellular Naϩ and Kϩ ions for the neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1; the rat homologue of human EAAT3). Our data show that external Kϩ is able to activate the glutamate transporter anion conductance, but only when extracellular Naϩ is present These results suggest the existence of a state in which Naϩ and Kϩ can bind simultaneously to the transporter, in contrast to previous hypotheses of these two cations acting in a competitive fashion [23]. A kinetic model to explain the data includes a novel “co-binding” state, suggesting that at least one cation-binding site is poorly selective for Naϩ versus Kϩ
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