Conformationally Sensitive Residues in Transmembrane Domain 9 of the Na+/dicarboxylate Co-transporter

Ana M Pajor

doi:10.1074/jbc.m011387200

Abstract

The Na(+)/dicarboxylate co-transporter, NaDC-1, couples the transport of sodium and Krebs cycle intermediates, such as succinate and citrate. Previous studies identified two functionally important amino acids, Glu-475 and Cys-476, located in transmembrane domain (TMD) 9 of NaDC-1. In the present study, each amino acid in TMD-9 was mutated to cysteine, one at a time, and the accessibility of the membrane-impermeant reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) to the replacement cysteines was determined. Cysteine substitution was tolerated at all but five of the sites: the A461C mutant was not present at the plasma membrane, whereas the F473C, T474C, E475C, and N479C mutants were inactive proteins located on the plasma membrane. Cysteine substitution of four residues found near the extracellular surface of TMD-9 (Ser-478, Ala-480, Ala-481, and Thr-482) resulted in proteins that were sensitive to inhibition by MTSET. The accessibility of MTSET to the four substituted cysteines was highest in the presence of the transported cations, sodium or lithium, and low in choline. The four mutants also exhibited substrate protection of MTSET accessibility. The MTSET accessibility to S478C, A481C, and A480C was independent of voltage. In contrast, T482C was more accessible to MTSET in choline buffer at negative holding potentials, but there was no effect of voltage in sodium buffer. In conclusion, TMD-9 may be involved in transducing conformational changes between the cation-binding sites and the substrate-binding site in NaDC-1, and it may also form part of the translocation pathway through the transporter.

Highlights

High and low affinity transporters for dicarboxylates and sulfate (1, 6)
Cysteine substitution of four residues found near the extracellular surface of transmembrane domain (TMD)-9 (Ser-478, Ala-480, Ala-481, and Thr-482) resulted in proteins that were sensitive to inhibition by MTSET
The results of this study show that cysteine replacement of four amino acids found near the extracellular surface of TMD-9 (Ser-479, Ala-480, Ala-481, and Thr-482) produces mutant transporters that are sensitive to inhibition by MTSET

Summary

EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagenesis—Site-directed mutagenesis was done using the method of Kunkel (13) with reagents from the Muta-gene kit from Bio-Rad, according to the manufacturers instructions. Preincubation of Oocytes with MTSET—Oocytes were washed three times with 3 ml of choline buffer and incubated at room temperature with 0.4 ml of MTSET (Toronto Research Biochemicals) in the appropriate buffer (for example, choline buffer or sodium buffer) for a time period up to 10 min. The MTSET-containing solution was removed with suction and the oocytes were washed four times with 3 ml of choline buffer at room temperature. The transport solution containing radioactive substrate was added and transport was measured as described above. Electrophysiology—Currents in oocytes expressing cysteine-substituted mutants of NaDC-1 were measured using the two-electrode voltage clamp method as described previously (5). In experiments testing different holding potentials, the oocyte holding potential was adjusted between 0 and Ϫ150 mV, the MTSET solution was superfused for a period of 10 –30 s, followed by a 2-min washout in the same buffer. The holding potential was returned to Ϫ50 mV before the voltage pulses were applied

RESULTS

TABLE I Succinate kinetics in cysteine substitution mutants

DISCUSSION