Substrate specificity and functional characterization of sodium/dicarboxylate cotransporters



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Transport of dicarboxylates across the plasma membrane is mediated by the Na+/dicarboxylate cotransporters (NaDCs) belonging to the SLC13 gene family. These transporters play important roles in the homeostasis of dicarboxylates. The studies in this dissertation focused on two aspects of the NaDCs: structure-function studies of two low-affinity transporters, mouse (m) and rabbit (rb) NaDC1, and functional characterization of a high-affinity NaDC transporter from Xenopus laevis, xNaDC3. \r\n\r\nAlthough sharing strong sequence identity, mNaDC1 and rbNaDC1 differ in their ability to transport certain dicarboxylates. For example, oocytes expressing mNaDC1 exhibit large inward currents in the presence of glutarate, adipate, and succinate, whereas oocytes expressing rbNaDC1 have currents only with succinate. To identify NaDC1 domains involved in different ability to transport glutarate and adipate, I constructed a series of mNaDC1-rbNaDC1 chimeras, and used both electrophysiological and dual-radiolabel competitive uptake techniques to exam their transport properties. My work indicates that different multiple transmembrane helices (TMs) are involved in NaDC1 substrate recognition, with the region of TM 3-4 and the C-terminus required for glutarate while the TM 8-10 region is necessary for adipate transport. Further analysis of these two regions provided evidence that they contained residues important for both apparent substrate affinity and catalytic efficiency of NaDC1.\r\n\r\nThe functional properties of non-mammalian vertebrates in the SLC13 family are not well characterized. Therefore, an initial functional characterization of xNaDC3 was performed using electrophysiological techniques. Like other members of the SLC13 family, xNaDC3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC3 are unique and involve large cation-activated leak currents possibly mediated by anions. \r\n\r\nTaken together, these studies have provided insight into the mechanism of substrate recognition and transport by NaDCs. My work not only contributes to a more detailed analysis of NaDC structure-function relationships, but also demonstrates how transport protein structural information can be obtained using a biochemical approach. The need for such an approach can be explained by the fact that only a limited number of transporters have had their structures solved to an atomic resolution despite the critical involvement of transporters in cellular functions. \r\n



Xenopus laevis oocytes, two-electrode voltage clamp, transport specificity ratio, succinate, glutarate, citric acid cycle intermediates, adipate