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

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.