Distinct roles of the Na⁺ binding sites in the allosteric coupling mechanism of the glutamate transporter homolog, Glt_Ph

Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na⁺ and substrate. The sodium coupled Asp transporter, Glt_Ph is an archaeal homolog of glutamate transporters that has been extensively used to probe the transport mechanism. Previous studies have shown that hairpin-2 (HP2) functions as the extracellular gate for the aspartate binding site and plays a key role in the coupled binding of sodium and aspartate to Glt_Ph. The binding sites for three Na⁺ ions (Na1-3) have been identified in Glt_Ph, but the specific roles of the individual Na⁺ sites in the binding process have not been elucidated. In this study, we developed assays to probe Na⁺ binding to the Na1 and Na3 sites and to monitor the conformational switch in the NMDGT motif. We used these assays along with a fluorescence assay to monitor HP2 movement and EPR spectroscopy to show that Na⁺ binding to the Na3 site is required for the NMDGT conformational switch while Na⁺ binding to the Na1 site is responsible for the partial opening of HP2. Complete opening of HP2 requires the conformational switch of the NMDGT motif and therefore Na⁺ binding to both the Na1 and the Na3 sites. Based on our studies, we also propose an alternate pathway for the coupled binding of Na⁺ and Asp.