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The sodium-dependent di- and tricarboxylate transporter, NaCT, is not responsible for the uptake of D-, L-2-hydroxyglutarate and 3-hydroxyglutarate into neurons.
Brauburger K
,
Burckhardt G
,
Burckhardt BC
.
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Concentrations of glutarate (GA) and its derivatives such as 3-hydroxyglutarate (3OHGA), D- (D-2OHGA) and L-2-hydroxyglutarate (L-2OHGA) are increased in plasma, cerebrospinal fluid (CSF) and urine of patients suffering from different forms of organic acidurias. It has been proposed that these derivatives cause neuronal damage in these patients, leading to dystonic and dyskinetic movement disorders. We have recently shown that these compounds are eliminated by the kidneys via the human organic anion transporters, OAT1 and OAT4, and the sodium-dependent dicarboxylate transporter 3, NaDC3. In neurons, where most of the damage occurs, a sodium-dependent citrate transporter, NaCT, has been identified. Therefore, we investigated the impact of GA derivatives on hNaCT by two-electrode voltage clamp and tracer uptake studies. None of these compounds induced substrate-associated currents in hNaCT-expressing Xenopus laevis oocytes nor did GA derivatives inhibit the uptake of citrate, the prototypical substrate of hNaCT. In contrast, D- and L-2OHGA, but not 3OHGA, showed affinities to NaDC3, indicating that D- and L-2OHGA impair the uptake of dicarboxylates into astrocytes thereby possibly interfering with their feeding of tricarboxylic acid cycle intermediates to neurons.
Fig. 1. Effects of GA derivatives on hNaCT-expressing oocytes. Current-voltage (I-V) relations of substrate-associated currents in hNaCT-expressing oocytes are shown in (a), (c) and mocks (b). Oocytes were superfused first with ORi and subsequently with ORi to which 1 mM of the respective GA derivative was added. Subtraction of the currents obtained in the presence from those in the absence of the respective substrate revealed the substrate-associated currents, ΔI. Oocytes were first tested for the current induced by the prototypical substrate, citrate, to test for successful expression and only oocytes showing citrate-associated currents >20 nA at −90 mV were used in this study. Afterwards, 3OHGA, D-, L-2OHGA, αKG, and GA were applied at random order. a, b Means ± SEM of 5 oocytes from 4 donors and of 3 oocytes from 3 donors, respectively. c Comparison of the uptake of labeled citrate and glutarate in the same batch of oocytes. Whereas the hNaCT-expressing oocytes (3 independent experiments with 10 oocytes for each experimental condition) took up citrate (12 μM), no uptake of glutarate (20 μM) was observed.
Fig. 2. Effect of succinate and glutamate on hNaDC3. a Current-voltage (I-V) relations as a function of membrane potential (Vc) in hNaDC3-expressing oocytes were obtained by substraction of the currents in the presence of 1 mM succinate in ORI (●) or of 1 mM glutamate (○) in ORI from those measured in ORI alone. Data present mean values ± SEM of 4 oocytes from 4 donors. b Succinate uptake in the absence and presence of 1 mM glutamate in hNaDC3-expressing oocytes (black columns) and mocks (grey columns) as obtained in two independent experiments with 8–10 oocytes for each experimental condition. Uptake of succinate in the absence and presence of glutamate was not significantly different.
Fig. 3. Michaelis-Menten constants (KM (mM)) (a) and maximal substrate-inducible currents (Imax (nA)) (b) of the GA derivatives as a function of membrane potential (Vc). hNaDC3-expressing oocytes were subsequently superfused with increasing substrate concentrations of 3OHGA: 0.1, 0.2, 0.5, 1, 2, and 5 mM; D- and L-2OHGA: 0.01, 0.05, 0.1, 0.5, and 1 mM in ORi at pH 7.5. Values were calculated using the SigmaPlot Systat program. Data were obtained at the end of a 10-s perfusion with the respective solution at the indicated Vc and were calculated from 7 oocytes of 3 donors for 3OHGA, for D-2OHGA from 3 oocytes from 3 donors, and for L-2OHGA from 5 oocytes from 4 donors.
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