Figure 1.
Structures of EAAT substrates and non-substrates.
Diagrams of relevant amino acids and the associated side chain pKas. Amino acids are depicted in their primary charge state at physiological pH. Abbreviations are: L-Asp, L-aspartate; L-Glu, L-glutamate; L-Sec, L-selenocysteine; L-Cys, L-cysteine; L-Ser, L-serine.
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).