Sodium-dependent extracellular accessibility of Lys-84 in the sodium/dicarboxylate cotransporter

Abstract

The Na(+)/dicarboxylate cotransporter transports Na(+) with citric acid cycle intermediates such as succinate and citrate. The present study focuses on transmembrane helix 3, which is highly conserved among the members of the SLC13 family. Fifteen amino acids in the extracellular half of transmembrane helix (amino acids 98-112) as well as Lys-84, previously shown to affect substrate affinity, were mutated individually to cysteine and expressed in the human retinal pigment epithelial cell line. Transport specificity ratio analysis shows that determinants for distinguishing succinate and citrate are found at amino acids Lys-84, Glu-101, Trp-103, His-106, and Leu-111. 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. The sensitivity of K84C to MTSES was greatest in the presence of sodium, and the inhibition could be prevented by addition of substrate or replacement of sodium, indicating that the accessibility of Lys-84 changes with conformational state. The substrate protection of MTSES inhibition of K84C appears to occur early in the transport cycle, before the large-scale conformational change associated with translocation of substrate. The results point to a new location for Lys-84 within the substrate access pore of the Na(+)/dicarboxylate cotransporter, either in a transmembrane helix or a reentrant loop facing a water-filled pore.

FIG. 1. Multiple sequence alignment of TM3 and connecting loops in members of the SLC13 family

The amino acid numbering (76–112) is based on the rbNaDC1 sequence. The sequence alignment was performed using the ClustalW program (default parameters, Gonnet matrix). Other members of the SLC13 family include the high affinity Na⁺/dicarboxylate cotransporters (NaDC3), Na⁺/citrate cotransporter (NaCT), Na⁺/sulfate cotransporters (NaS) and the Drosophila dicarboxylate exchanger (Indy). The Genbank^TM accession numbers for nucleotide sequences are next to the names. Positions of conserved amino acid residues in the proteins are highlighted in grey. Amino acids in rbNaDC1 mutated in this study are indicated by * above the sequence. The amino acids before the predicted TM3 sequence are part of the intracellular loop connecting TM2 and 3.

FIG. 2. Western blots of (A) cell-surface and (B) total protein expression

HRPE cells expressing cysteine mutants were treated with Sulfo-NHS-LC biotin as described in “Experimental Procedures”. The upper panel shows the cell surface protein expression, and the lower panel shows the intracellular protein expression. Western blots were probed with anti-NaDC1 antibodies (1:1000 dilution). Each blot includes an internal control of the parental transporter, C476S. The positions of the molecular mass standard (MagicMark XP) are indicated on the left. Two immunoreactive bands at approximately 70 and 55 kDa represent differently glycosylated forms of rbNaDC1 (15).

FIG. 3. Transport activity and protein expression of cysteine-substituted mutants

The data are shown as a percentage of the C476S control from the same blot or uptake experiment. Transport of 100 μM [³H]succinate was measured with 30 min incubation in sodium containing buffer. Transport results shown are mean ± S.E.M. (n = 3–11). Protein abundance was determined by quantitating the intensities of NaDC1 protein bands from Western blots, such as those shown in Fig. 2. The bars represent mean ± range or S.E.M. (n = 2–4 blots, separate transfections).

FIG. 4. Dual-label competitive transport of cysteine-substituted NaDC1 mutants

(A) Competitive uptake of [³H]succinate (10 μM) and [¹⁴C]citrate (20 μM) by cysteine-substituted mutants. The transport assay was performed for 20 min. (B) Transport specificity ratios (TSR succinate: citrate) of NaDC1 mutants, calculated from data shown in (A). Bars are mean ± S.E.M. (n = 4). * p < 0.05, significantly different from C476S control group.

FIG. 5. Effect of MTSES on succinate transport by cysteine-substituted NaDC1 mutants

HRPE cells expressing mutants were preincubated with 1 mM MTSES in sodium buffer or with sodium buffer alone (control) for 20 min. The [³H]succinate uptake activity remaining after the preincubation was then measured. Uptake activities in cells pretreated with MTSES are expressed as a percentage of the uptakes in cells preincubated with sodium buffer alone. Data shown are means ± S.E.M., n = 3 experiments. * p< 0.05, significantly different from control group for that mutant.

FIG. 6. Concentration dependence of MTSES inhibition of succinate transport by K84C mutant

HRPE cells were preincubated with increasing concentration of MTSES in sodium buffer. The succinate transport activity was measured and expressed as a percentage of the transport activity in the control group treated with sodium buffer alone. Each point represents the mean ± range (n = 2) from a single experiment.

FIG. 7. Cation, substrate and temperature dependence of MTSES labeling

(A) HRPE cells transiently expressing the K84C mutant or the pcDNA3.1 vector were preincubated 20 min at room temperature with or without 10 μM MTSES. The preincubation buffers contained sodium or choline, with or without 10 mM succinate. The preincubation solution was washed away, and the remaining uptake activity with [³H]succinate was then measured, as described previously. Uptake activities of cells pretreated with MTSES are shown as a percentage of the uptake in control cells pretreated with the same buffer without MTSES. Data shown are mean ± S.E.M., n = 3 experiments. * p< 0.05, significant difference compared with control group. (B) Effect of temperature on substrate protection. HRPE cells transiently expressing the K84C mutant were preincubated in sodium buffer with increasing concentrations of succinate (0–1000 μM) and with or without 10 μM MTSES. The preincubations were done at room temperature (RT) or on ice for 20 min. After pretreatment, the preincubation solution was washed away and the remaining uptake activity of [³H]succinate was measured at room temperature. Data shown are mean ± range, n = 2. The half-saturation constant was calculated using non-linear regression to ν= (Vmax * [S])/(K_0.5 + [S]) + C, where C represents the transport activity remaining after MTSES treatment in the absence of succinate. C is 26% at room temperature and 35% on ice.

FIG. 8. Labeling of cysteine-substituted NaDC1 mutants with MTSEA-biotin

(A) HRPE cells expressing K84C, I98C or R112C mutants were initially preincubated in sodium buffer with (+) or without (−) 1 mM MTSES in order to label the accessible cysteines. The preincubation solution was washed away and the cell monolayers were then incubated with MTSEA-biotin and transferred to Western blots as described in “Experimental Procedures”. The negative control mutant, C476S, and the positive control, T482C, were included in each biotinylation experiment. The positions of chemiluminescent size standards, MagicMark XP (Invitrogen), are shown in the leftmost lane. (B) Comparison of the band intensity analysis of MTSEA-biotinylation. Western blots such as those shown in (A) were quantitated and expressed as a percentage of the T482C intensity from the same blot. The difference between the signal in the presence and absence of MTSES represents specific binding of MTSEA-biotin. The bars represent mean ± range (n = 2 blots, separate transfections).

FIG. 9. Models summarizing results of cysteine-scanning mutagenesis experiments in NaDC1

Two alternative topology models to explain the results are shown in A and B. In model A, Lys-84 is located within the TM3 and in model B, Lys-84 is accessible to the outside of the cell as part of a reentrant loop. K84C is sensitive to inhibition by MTSES, is labeled by MTSEA-biotin and shows substrate protection of labeling. R112C, shown as a dark grey circle, is labeled by MTSEA-biotin but is insensitive to inhibition by MTSES. Residues that showed changes in transport specificity ratio (TSR) are indicated by bold circles and the positions of inactive cysteine-substituted mutants are shown by grey circles with dashed lines.