Molecular transport properties of sodium/dicarboxylate cotransporters

The Na+/dicarboxylate cotransporter (NaDC1) plays important roles in absorption of citric acid cycle intermediates from the intestinal and renal tubular lumen. NaDC1 influences the homeostasis of citrate, which may be associated with the formation of kidney stones. The studies in this dissertation focused on two aspects of NaDC1: the transport pathway of di-and tricarboxylates in the small intestine and structure-function studies of NaDC1. \r\nThe transport pathways of di-and tricarboxylates in the intestine have not been clearly identified; thus, the identification of intestinal di-and tricarboxylate transport pathway was investigated in human Caco-2 cell line. The results show that these cells contain at least three distinct transporters, including the Na+-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and a sodium-independent pathway, possibly an organic anion transporter. Most of the succinate transport is mediated by sodium-dependent pathways, predominantly NaDC1. In contrast, citrate transport in Caco-2 cells occurs by a combination of sodium-independent pathways, possibly mediated by an organic anion transporter, and sodium-dependent mechanisms. \r\nThe amino-terminal half of the protein in NaDC1, in particular transmembrane helix (TM) 3, appears to be important in determining substrate specificity and affinity; therefore, the investigation of the permeation pathway in NaDC1 was examined. The extracellular half of TM3 in NaDC1 was studied using the substituted-cysteine accessibility method and the transport specificity ratio (TSR). The TSR analysis provides evidence that TM3 contains determinants for substrate specificity and catalytic efficiency. All of the mutants were tested for sensitivity to the membrane-impermeant cysteine-specific reagent (2-sulfonatoethyl) methanethiosulfonate (MTSES), but only K84C was sensitive to MTSES inhibition. Pre-exposure of K84C to succinate results in protection from MTSES inhibition. The mechanism of substrate protection appears to be steric hindrance, rather than large-scale conformational change. The data suggest that TM3 may be located in the permeation pathway and also point to a new location for Lys-84. \r\nThe interaction between TM7, 10 and 11 of NaDC1 contribute to the differences in substrate and cation affinity between rabbit (rb) and human (h) NaDC1. However, the important residues responsible for the differences have not been identified. The identification of residues was focused in the TM 10 region. Four mutants were made in which the rabbit sequence was substituted for that of the human NaDC1. The human-T509S mutant transporter exhibits the cation and substrate affinity and specificity of the R10 chimera, hNaDC1 with a substitution of TM10 and associated loop from rbNaDC1. The rabbit-S512T mutation made in rbNaDC1 at the equivalent position has properties similar to those of human NaDC1. It appears that a serine or threonine at position 509 (in human NaDC1) or 512 (in rabbit NaDC1) determines functional differences between NaDC1 orthologs in both substrate and cation transport. \r\nNaDC1 is implicated in many physiological processes, it is essential to understand its structure and function. These studies have provided new fundamental structural-function relationship information on the transport mechanism of NaDC1. As membrane proteins are one of the key components for drug designing and targeting, this knowledge will provide information in the development of novel therapies with NaDC1 as a target. \r\n