Mapping Functionally Important Residues in the Na+/Dicarboxylate Cotransporter, NaDC1

Abstract

Transporters from the SLC13 family couple the transport of two to four Na⁺ ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na⁺/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue VcINDY in an inward-facing conformation with one sodium ion bound at the Na1 site. In the study presented here, we modeled the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1, respectively) using an outward-facing model of VcINDY as a template and identified residues in or near the putative Na2 and Na3 cation binding sites. Guided by the structural models in both conformations, we performed site-directed mutagenesis in rbNaDC1 for residues proposed to be in the Na⁺ or substrate binding sites. Cysteine substitution of T474 in the predicted Na2 binding site results in an inactive protein. The M539C mutant has a low apparent affinity for both sodium and lithium cations, suggesting that M539 may form part of the putative Na3 binding site. The Y432C and T86C mutants have increased K_m values for succinate, supporting their proposed location in the outward-facing substrate binding site. In addition, cysteine labeling by MTSEA-biotin shows that Y432C is accessible from the outside of the cell, and the accessibility changes in the presence or absence of Na⁺. The results of this study improve our understanding of substrate and ion recognition in the mammalian members of the SLC13 family and provide a framework for developing conformationally specific inhibitors against these transporters.