Role of isoleucine-554 in lithium binding by the Na+/dicarboxylate cotransporter NaDC1

Sodium-coupled transport of citric acid cycle intermediates, such as succinate and citrate, is mediated by the NaDC1 transporter located on the apical membrane of kidney proximal tubule and small intestine cells. Our previous study showed that transmembrane helix (TM) 11 of NaDC1 is important for sodium and lithium binding, as well as for determining citrate affinity [Kahn and Pajor (1999) Biochemistry 38, 6151]. In the present study, 21 amino acids in TM11 and the extracellular loop of NaDC1 were mutated one at a time to cysteine. All of the mutants were well expressed on the plasma membrane, but many of them had decreased transport activity. The G550C, W561C, and L568C mutants were inactive, suggesting that these residues may be critical for function. None of the cysteine mutants was sensitive to inhibition by the membrane-impermeant cysteine reagents, MTSET or MTSES, suggesting that the helix is inaccessible to the extracellular solvent. Although NaDC1 is inhibited by low concentrations of lithium in the presence of sodium, the I554C mutant was stimulated by lithium with a K(0.5) of 4.8 mM. The I554C mutant also had decreased affinity for sodium. We conclude that TM11 is likely to be an outer helix in NaDC1 that contains several residues critical for transport. Ile-554 in the middle of the helix may be an important determinant of cation affinity and selectivity, in particular the high affinity cation binding site that recognizes lithium.