Analysis of naturally occurring mutations in the human uptake transporter NaCT important for bone and brain development and energy metabolism

Abstract

The human uptake transporter NaCT is important for human brain development, brain function and energy metabolism and mediates the uptake of citrate and other intermediates of the tricarboxylic acid cycle from blood into neurons and hepatocytes. Mutations in the SLC13A5 gene encoding NaCT are associated with epileptic encephalopathy. To gain more insights into the transport mechanisms we analyzed the functional consequences of mutations in the SLC13A5 gene on NaCT-mediated transport function. Using HEK293 cells expressing wild-type and eight mutated NaCT proteins, we investigated the mRNA and protein amount as well as the protein localization of all NaCT variants. Furthermore, the impact on NaCT-mediated citrate uptake was measured. In addition, a structural model of the transport pore was generated to rationalize the consequences of the mutations on a structural basis. We demonstrated that all proteins were synthesized with an identical molecular weight as the wild-type transporter but several mutations (NaCTp.G219R, -p.G219E, -p.T227M, -p.L420P and -p.L488P) lead to a complete loss of NaCT-mediated citrate transport. This loss of transport activity can be explained on the basis of the developed structural model. This model may help in the further elucidation of the transport mechanism of this important uptake transporter.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Transmembrane model of human NaCT with the localization of the respective mutant amino acids investigated in this study.

Figure 2

Characterization of transfected HEK293 cells expressing wild-type and mutated NaCT proteins. Characterization of SLC13A5 mRNA expression (A) and of the NaCT protein amount in total membrane fractions (B) and in plasma membrane fractions (C) in transiently-transfected HEK293 cells. (A) Parental HEK293 cells were transiently-transfected with the expression plasmids encoding the wild-type NaCT transporter (wild-type) or with expression plasmids encoding variants of the NaCT protein. Parental HEK293 cells transfected with the empty expression vector served as control (control). All expression values are given in percent ± SD of the respective ß-actin mRNA expression measured under the same experimental conditions. (B) Immunoblot analysis of total membrane fractions of transiently-transfected HEK293 cells expressing different variants of the NaCT protein. Membrane isolation has been verified by staining the membrane protein pan-cadherin. Presented are the merged pictures of two immunoblots conducted under identical conditions. (C) Immunoblot analysis of plasma membrane fractions of transiently-transfected HEK293 cells expressing different variants of the NaCT protein. Membrane isolation has been verified by staining the membrane protein pan-cadherin. Presented are the merged pictures of two immunoblots conducted under identical conditions. The original immunoblots detecting NaCT in the different transient transfections are shown in the supplement (Supplement Figs 1 and 2).

Figure 3

Localization of wild-type and mutated NaCT proteins. Immunofluorescence analysis of NaCT protein localization in transiently-transfected HEK293 cells expressing wild-type or variant NaCT proteins. Empty vector-transfected HEK293 cells served as control.

Figure 4

Citrate uptake mediated by wild-type and mutated NaCT proteins. Uptake of citrate (1 µM) into transiently-transfected HEK293 cells. Parental HEK293 cells were transiently-transfected and citrate uptake was measured under established experimental conditions. Cells were transfected with expression plasmids containing cDNAs encoding the wild-type NaCT protein (wild-type) or different mutant NaCT proteins. Citrate net uptake mediated by the wild-type NaCT protein was set to 100% and all net uptake values are given in relation to this uptake ± SD. ***P 

Figure 5

Molecular model of the human NaCT protein and localization of amino acids investigated in this study. (A) Molecular model of human NaCT indicating sites of investigated mutations. One of the subunits is shown as grey ribbon and the other subunit is shown in color gradient ranging from blue (N-terminus) to red (C-terminus). In both subunits, a citrate molecule is shown in stick presentation (atom-type coloring). The grey subunit additionally shows the position of the residues investigated by side-directed mutagenesis. These residues are shown in space-filled presentation using the following color code: G219 (yellow), T227 (orange), D243 (cyan), L420 (dark blue), L485 (red), L488 (green) and F500 (light blue). (B) Same presentation as in (A) but rotated by 90° around the horizontal axis. This corresponds to a view on the NaCT protein from the extracellular site. (C,D) Effect of the T227M mutation on the NaCT structure. In the wild-type protein, T227 (orange) is close to the substrate citrate (atom-type coloring). (D) Due to its longer sidechain M227 (orange) would form clashes with a citrate, which will no longer allow the transport of this substrate. (E,F) Effect of the G219R mutation on the NaCT structure. In the wild-type protein G219 (yellow) interacts with A133 (atom-type coloring) of an adjacent helix. (F) Due to its longer sidechain R219 (yellow) would form clashes with A133 thereby disrupting the NaCT structure.

Figure 6

Clustal analysis of NaCT proteins from different species and of members of the SLC13 family. (A) For the NaCT clustal analysis of different species the sequences NM_001143838.2 (human), HM998308.1 (chimpanzee), HM998307.1 (macaque), KF728381.1 (pig), NM_001004148.4 (mouse) and HM998309.1 (zebra fish) were used. (B) For the NaCT clustal analysis of the human SLC13 family members the sequences NM_001143838.2 (NaCT), NM_003984.3 (NaDC2), NM_022829.5 (NaDC3), NM_022444.3 (NaSI1) and NM_012450.2 (NaSUT1) were used. Clustal analysis was performed using the HUSAR program package from the German Cancer Research Center. All investigated mutations are indicated as boxes.