Watts SD et al. (2014), Cysteine transport through excitatory amino aci...

XB-ART-49660

PLoS One 2014 Jan 01;910:e109245. doi: 10.1371/journal.pone.0109245.

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Cysteine transport through excitatory amino acid transporter 3 (EAAT3).

Watts SD , Torres-Salazar D , Divito CB , Amara SG .

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Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.

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Species referenced: Xenopus
Genes referenced: slc1a1 slc1a2 slc1a3 slc1a4 sult2a1

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	Figure 2. Selenocysteine is transported by EAATs 1–3.A) Representative recordings (upper panel) and averaged normalized transport currents (lower panel) measured at −60 mV as a function of the L-selenocysteine concentration in EAAT3 expressing oocytes (n = 6). Data are presented as the mean and Std. dev. of the mean and fit with the Hill equation to estimate the Km for transport. B) Comparison of the maximal transport currents at −60 mV for L-selenocysteine and L-cysteine by EAAT1 (n>3), EAAT2 (n>5) or EAAT3 (n>10) normalized to the maximal currents induced by L-glutamate measured in the same oocyte. C) Comparison of averaged current-voltage relationships recorded from oocytes expressing EAAT3 for both 1 mM glutamate (red symbols, n = 4) and 1 mM selenocysteine (blue symbols, n = 4). Black symbols indicate the averaged current voltage relationship of the same cells in the absence of substrate (n = 4) and the solid line represents the average of water injected oocytes in the presence of 1 mM glutamate (n = 5).
	Figure 3. Inhibition of glutamate transport by L-Selenocysteine and L-Cysteine for EAAT 2 and EAAT 3.Inhibition of radiolabeled glutamate uptake using varying concentrations of L-cysteine (circles) or L-selenocysteine (triangles) in HEK293 cells expressing EAAT2 (A, n>5 for each data point) or EAAT3 (B, n>5 for each data point). Data are represented as the mean and the standard error of the mean with non-linear curve fit to calculate the IC50s.
	Figure 4. pH affects glutamate inhibition of cysteine transport.Glutamate inhibition of cysteine uptake from oocytes expressing EAAT3 at three different cysteine concentrations: 30 µM (circles), 300 µM (triangles) and 1 mM (squares) at pH 6.9 (A) and pH 8.5 (B).
	Figure 5. mEGFPpH detects intracellular pH changes induced by glutamate transport.Representative image of mEGFPpH transfected HEK293 cells (A) and representative fluorescence traces from HEK293 cells expressing mEGFPpH perfused with 50 mM NH4Cl (B). Y axis indicates the ratio of fluorescence emission at 510 nm from excitation at 485 nm and 405 nm (F485/F405) (B). Arrows in A indicate the cells from which the traces in B were recorded. C) Fluorescence ratio (F485/F405) as a function of induced intracellular pH following NH4Cl perfusion (B). D) Perfusion of increasing concentrations of L-glutamate results in increased rate of mEGFPpH fluorescence decrease in HEK293 cells co-transfected with EAAT3 and mEGFPpH. The Y-axis units are the fluorescence ratio for emission at 510 nm with excitation at 485 and 405 nm (F485/F405). E) Perfusion with 100 µM D-aspartate results in intracellular acidification with slope magnitude similar to that for 100 µM L-glutamate (bar graph). Y-axis units are the fluorescence ratio for emission at 510 nm with excitation at 485 and 405 nm (F485/F405). F) Representation of the magnitude of the slope of mEGFPpH fluorescence ratio decrease (left y-axis) as a function of the applied glutamate concentration compared with the glutamate transport activity (right y-axis) in similarly transfected cells.
	Figure 6. Transport of substrates in cells expressing EAAT2 or EAAT3 differentially affects intracellular pH.Representative fluorescence recordings from HEK293 cells expressing EAAT2 (A) or EAAT3 (B) in response to short applications of different concentrations of L-cysteine, L-glutamate or L-selenocysteine. The magnitude of maximal steady state slopes (A and B) are plotted in bar graphs below each trace, normalized to the glutamate slope magnitude. C) Representative trace of the effect on cysteine induced mEGFPpH fluorescence changes in EAAT3 expressing HEK293 cells with (left) or without (right) 100 µM TBOA. The Y-axis units for traces represent the fluorescence ratio for emission at 510 nm with excitation at 485 and 405 nm (F485/F405).
	Figure 7. EAAT3 dependent release of [3H]-L-glutamate or [35S]-L-cysteine.A and B) Release of [35S]-L-Cysteine (A) or [3H]-L-Glutamate (B) from oocytes co-expressing EAAT3 and ASCT, in response to different buffers and conditions C) Averaged current-voltage relationships recorded from oocytes expressing EAAT3 alone (n = 6) or co-expressed with ASCT1 (n = 4) in response to a family of voltage pulses in the absence (black symbols) and the presence of 1 mM serine (blue symbols) or 1 mM glutamate (red symbols). The solid line represents un-injected oocytes in the presence of 1 mM glutamate and 1 mM serine (n = 3).

References [+] :

Amara, Excitatory amino acid transporters: keeping up with glutamate. 2002, Pubmed