A K+/Na+ co-binding state: Simultaneous versus competitive binding of K+ and Na+ to glutamate transporters

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.