Mechanism and potential sites of potassium interaction with glutamate transporters

In the mammalian glutamate transporters, countertransported intracellular K(+) is essential for relocating the glutamate binding site to the extracellular side of the membrane. This K(+)-dependent process is believed to be rate limiting for the transport cycle. In contrast, extracellular K(+) induces glutamate release upon transporter reversal. Here, we analyzed potential K(+) binding sites using molecular dynamics (MD) simulations and site-directed mutagenesis. Two candidate sites were identified by spontaneous K(+) binding in MD simulations, one site (K1 site) overlapping with the Na1 Na(+) binding site and the K2 site being localized under hairpin loop 2 (HP2). Mutations to conserved amino acid residues in these sites resulted in several transporters that were defective in K(+)-induced reverse transport and which bound K(+) with reduced apparent affinity compared with the wild-type transporter. However, external K(+) interaction was abolished in only one mutant transporter EAAC1(D454A) in the K1 site. Our results, for the first time, directly demonstrate effects of K1-site mutations on K(+) binding, in contrast to previous reports on K(+) binding sites based on indirect evidence. We propose that K(+) binding to the K1 site is responsible for catalyzing the relocation step, whereas binding to the K2 site may have an as-of-yet unidentified regulatory function.