Mutations in the Na(+)/citrate cotransporter NaCT (SLC13A5) in pediatric patients with epilepsy and developmental delay

Abstract

Mutations in the SLC13A5 gene that codes for the Na(+)/citrate cotransporter, NaCT, are associated with early onset epilepsy, developmental delay and tooth dysplasia in children. In the present study we identify additional SLC13A5 mutations in nine epilepsy patients from six families. To better characterize the syndrome, families with affected children answered questions about the scope of illness and treatment strategies. There are currently no effective treatments, but some anti-epileptic drugs targeting the GABA system reduce seizure frequency. Acetazolamide, a carbonic anhydrase inhibitor and atypical anti-seizure medication decreases seizures in 4 patients. In contrast to previous reports, the ketogenic diet and fasting produce worsening of symptoms. The effects of the mutations on NaCT transport function and protein expression were examined by transient transfections of COS-7 cells. There was no transport activity from any of the mutant transporters, although some of the mutant transporter proteins were present on the plasma membrane. The structural model of NaCT suggests that these mutations can affect helix packing or substrate binding. We tested various treatments, including chemical chaperones and low temperatures, but none improve transport function in the NaCT mutants. Interestingly, coexpression of NaCT and the mutants results in decreased protein expression and activity of the wild-type transporter, indicating functional interaction. In conclusion, our study has identified additional SLC13A5 mutations in patients with chronic epilepsy starting in the neonatal period, with the mutations producing inactive Na(+)/citrate transporters.

Keywords: citrate; epilepsy; sodium; tooth hypoplasia; transporter.

Figure 1.

Secondary structure model of NaCT showing location of mutations in this study. The 11 transmembrane helices are shown as numbered rectangles and the opposing hairpin loops are labeled HP. The outside of the cell is at the top of the image.

Figure 2.

Transport activity of hNaCT wild-type and mutant transporters expressed in COS-7 cells. ¹⁴C-citrate transport (100 μmol/L) was measured at 37°C for 30 min. Data are means ± SEM, n = 8 (pcDNA and G219R), n = 7 (wild-type NaCT), n = 5 (DelG) and n = 3 (all others). * denotes p < 0.05 relative to pcDNA (empty vector plasmid) group.

Figure 3.

Western blots. (A) Total cell protein. Protein lysates from COS-7 cells transfected with empty vector, pcDNA, wild-type NaCT (WT) and mutants. Duplicate blots were probed with anti-NaCT (top) and GAPDH antibodies (bottom). Size standards (kDa) are shown at left. (B) Cell surface biotinylated proteins from HEK-293 cells treated with membrane-impermeant sulfo-NHS-LC-biotin followed by streptavidin beads. The HEK-293 cells were used for biotinylations because the signal was stronger than with COS-7 cells.

Figure 4.

Characterization of substrate transport by mutant and wild-type SLC13 transporters. Transport of ¹⁴C-citrate, ¹⁴C-succinate and ¹⁴C-glutarate (100 μmol/L each) in sodium or choline buffers. (A) DelG mutant. Bars show mean and range, n = 2 experiments. (B) G219R mutant. Bars show means ± SEM, n = 3–4; range is shown for n = 2 (succinate, glutarate with choline). (C) Comparison of citrate, succinate and glutarate transport in wild-type NaCT, NaDC1 and NaDC3 transporters. Transport was measured in sodium buffer. Bars show means ± SEM, n = 3 replicates from single experiment. In all experiments, backgrounds were corrected by subtracting counts in pcDNA-transfected cells. Note the 25-fold difference in scale between (C) and (A), (B).

Figure 5.

Effects of cotransfection of wild-type NaCT with vector plasmid (pcDNA) or mutant transporters. (A) Citrate transport activity. All groups were transfected with the same amount of wild-type NaCT DNA and a second plasmid. Activity is expressed as a percentage of the NaCT + pcDNA control group. All counts were corrected for background counts determined from cells transfected only with pcDNA plasmid. Bars are means ± SEM, n = 3–5, or range, n = 2 (DelG). * denotes p < 0.05 relative to the NaCT + pcDNA group. (B) Western blots. Duplicate blots were probed with anti-NaCT (top) and anti-GAPDH (bottom) antibodies. Groups were the same as for panel (A). The samples in the lane next to the size standards (labeled none) were transfected only with pcDNA, the other samples had two plasmid DNAs (NaCT and a second plasmid). All cells were transfected with the same total amount of DNA.

Figure 6.

Homology model of hNaCT showing the location of the point mutations. (A) Y82 is located in TM4, (B) G219 (TM5a), (C) T227 (TM5b) and (D) L488 and L492 (both located in TM10). In all panels, the transmembrane helices are shown as cyan cartoons; key amino acids represented by sticks with the mutated residues colored in orange. The purple sphere in panels (B) and (C) represents the sodium ion that binds the Na1 binding site. Citrate is shown in green sticks in panel (C).