It Runs in the Family: Determining the Transport Mechanism of Sodium/Dicarboxylate Symporter hNaDC3

Abstract

Members of the divalent anion:Na(+) symporter (DASS) family play important roles in mammalian physiology, transporting divalent anions, including Krebs cycle intermediates and sulfate, across the plasma membrane. These transporters may be key contributors to determining urinary citrate levels, which, in turn, may affect kidney stone formation; they also have been implicated in metabolic regulation in both drosophila and mammals. It is therefore important to understand the relationships in these proteins between their structure and functional mechanisms. Though no structures are yet available for mammalian DASS family members, a crystal structure of a bacterial homolog, vcINDY, has been determined. We recently demonstrated that vcINDY, a Na+-coupled succinate transporter, utilizes a dramatic “elevator” mechanism to transport substrate, involving a large-scale vertical movement of a protein domain perpendicular to the plane of the lipid bilayer membrane. Here, we sought to determine whether a mammalian family member utilizes a similar transport mechanism, specifically human NaDC3. With ∼30% identity between the vcINDY and hNaDC3, we hypothesized that a similar mechanism could be responsible for the functioning of hNADC3. Homology modeling with vcINDY suggested that several pairs of residues are brought into proximity upon substrate translocation. We prepared a series of double mutants introducing cysteines at positions predicted to be brought together in the outward-facing state of the protein and expressed them in Xenopus laevis oocytes. Using two-electrode voltage clamp and disulfide cross-linking, we investigated whether inhibition of transport activity observed upon formation of disulfides is consistent with the proposed mechanism of transport.