Cater RJ et al. (2016), Tuning the ion selectivity of glutamate transpo...

XB-ART-54360

J Gen Physiol 2016 Jul 01;1481:13-24. doi: 10.1085/jgp.201511556.

Show Gene links Show Anatomy links

Tuning the ion selectivity of glutamate transporter-associated uncoupled conductances.

Cater RJ , Vandenberg RJ , Ryan RM .

???displayArticle.abstract???
The concentration of glutamate within a glutamatergic synapse is tightly regulated by excitatory amino acid transporters (EAATs). In addition to their primary role in clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl(-) conductance. This conductance is activated by the binding of substrate and Na(+), but the direction of Cl(-) flux is independent of the rate or direction of substrate transport; thus, the two processes are thermodynamically uncoupled. A recent molecular dynamics study of the archaeal EAAT homologue GltPh (an aspartate transporter from Pyrococcus horikoshii) identified an aqueous pore at the interface of the transport and trimerization domains, through which anions could permeate, and it was suggested that an arginine residue at the most restricted part of this pathway might play a role in determining anion selectivity. In this study, we mutate this arginine to a histidine in the human glutamate transporter EAAT1 and investigate the role of the protonation state of this residue on anion selectivity and transporter function. Our results demonstrate that a positive charge at this position is crucial for determining anion versus cation selectivity of the uncoupled conductance of EAAT1. In addition, because the nature of this residue influences the turnover rate of EAAT1, we reveal an intrinsic link between the elevator movement of the transport domain and the Cl(-) channel.

???displayArticle.pubmedLink??? 27296367
???displayArticle.pmcLink??? PMC4924932
???displayArticle.link??? J Gen Physiol

Species referenced: Xenopus laevis
Genes referenced: slc1a3

???attribute.lit??? ???displayArticles.show???

	Figure 1. Structural conservation of an arginine residue at the interface of the transport domain and trimerization domain. (A) Cartoon representation of a locked R276S/M395R GltPh protomer in the plane of the membrane in the outward-facing occluded state (PDB accession no. 4X2S). The trimerization domain (TM1, TM2, TM4, and TM5) is colored in light brown, and the transport domain (TM3, TM6, TM7, TM8, HP1, and HP2) is colored in light blue, with HP1 and HP2 highlighted in yellow and red, respectively. In this mutant GltPh structure, the arginine has been moved from the native position of 276 to 395 to reflect the position of this positively charged residue in the EAATs. Residues 276 and 395 are shown in green stick representation and numbered according to GltPh positioning, with EAAT1 numbering provided in parentheses. Na+ ions are represented as purple spheres. Aspartate is not present in this structure. The figure was made using the software PyMOL (The PyMOL Molecular Graphics System, version 1.3r1; Schrödinger, LLC). (B) Amino acid alignment of HP1 and TM8 of the SLC1 family. R276/S363 and M395/R477 are outlined in red.
	Figure 2. R477H presents strong pH dependence for apparent glutamate affinity. (A–C) Representative current traces elicited by K0.5 concentrations (see Table 1) of l-glutamate for control (uninjected oocytes; A), WT EAAT (B), and R477H (C) at pH 5.5 (black bars) and 8.5 (red bars). (D and E) l-Glutamate concentration responses for WT EAAT1 (D) and R477H (E) were conducted at −60 mV in ND96 at pH 5.5 (black), 6.5 (orange), 7.5 (blue), and 8.5 (red). (F) Na+ concentration responses were performed at −60 mV for WT EAAT1 (closed squares) and R477H (open circles) at pH 5.5 in the presence of 300 µM or 3 µM l-glutamate for WT EAAT1 or R477H, respectively. Choline+ was used as the substitute cation. Currents were normalized to the maximal current activated for each cell. Data shown represent the mean ± SEM (n ≥ 3). Where error bars cannot be seen, they lie within the symbol.
	Figure 3. R477H presents strong pH dependence for substrate transport. Uptake of l-[3H]glutamate into oocytes expressing WT EAAT1 (A) and R477H (B) was measured at pH 5.5 (black), 6.5 (orange), 7.5 (blue), and 8.5 (red) over a course of 10 min. Oocytes expressing WT EAAT1 were incubated in 10 µM l-[3H]glutamate, and equivalent concentrations of l-[3H]glutamate were used for oocytes expressing R477H to account for the shifts in K0.5 for glutamate observed in this mutant transporter. See Materials and methods for further details. (C and D) Uptake of l-[3H]glutamate, into oocytes expressing WT EAAT1 (squares) and R477H (circles), was measured over a 120-min time period at pH 8.5 (red) and pH 5.5 (black). 10 µM l-[3H]glutamate was used for WT EAAT1 at both pH 5.5 and 8.5 and R477H at pH 8.5. 0.2 µM l-[3H]glutamate was used for R477H at pH 5.5 to account for differences in substrate affinity. (D) Uptake for l-[3H]glutamate into oocytes expressing R477H at pH 5.5 was lower than at pH 8.5 but is significantly above that of uninjected oocytes. A and B represent l-[3H]glutamate of WT EAAT1– and R477H-expressing oocytes; background uptake is presented in C and D. Background refers to l-[3H]glutamate uptake into uninjected oocytes for each condition. Data shown represent the mean ± SEM (n ≥ 7). Where error bars cannot be seen, they lie within the symbol. Note the scale difference in the y axes.
	Figure 4. The substrate-binding site is unperturbed in R477H at pH 5.5. (A) Steady-state currents at 60 mV in the presence of increasing concentrations of the competitive blocker TBOA were measured in WT EAAT1 and the R477H mutant transporter at pH 5.5. These currents were then subtracted from currents in the absence of TBOA to measure the TBOA-dependent blockage of the Na+-activated anion conductance. Currents were normalized to the maximal current blocked by TBOA for each cell. (B) l-Glutamate concentration responses for WT EAAT1 (closed squares) and R477H (open circles) at pH 5.5 reproduced from Fig. 2 are provided for comparison with [TBOA] concentration responses. (C) Current-voltage relationships for WT EAAT1 (closed squares) and R477H (open circles) as blocked by 30 µM TBOA at pH 5.5. Data shown represent the mean ± SEM (n ≥ 4). Where error bars cannot be seen, they lie within the symbol.
	Figure 5. pH-dependent uncoupled cation fluxes through R477H. (A–D) Current-voltage relationships for WT EAAT1 and R477H elicited by submaximal l-glutamate in 96 mM Cl−-based buffer (A and C) and 96 mM gluconate-based buffer (B and D) at pH 5.5 (black circles), 6.5 (orange triangles), 7.5 (blue triangles), and 8.5 (red squares). (E) Current at 60 mV for WT EAAT1 and R477H at pH 5.5 (black) and 8.5 (red) in 96 mM gluconate-based buffer (open bars) and 96 mM Cl−-based buffer (closed bars). Data shown represent the mean ± SEM (n ≥ 4). Where error bars cannot be seen, they lie within the symbol. Note the scale difference in the y axes.
	Figure 6. Na+ can permeate through R477H at pH 8.5. (A–D) Current-voltage relationships for WT EAAT1 and R477H elicited by submaximal l-glutamate in 10 mM (red squares) and 100 mM (blue squares) Na+-based buffers at pH 5.5 (A and C) and 8.5 (B and D). All buffers were osmotically balanced using choline+ as the substitute cation. Data shown represent the mean ± SEM (n ≥ 4). Where error bars cannot be seen, they lie within the symbol. Note the scale difference in the y axes.
	Figure 7. Cl− cannot permeate through R477H at pH 8.5. (A–D) Current-voltage relationships for WT EAAT1 and R477H elicited by submaximal l-glutamate in 10 mM (red squares) and 100 mM (blue squares) Cl−-based buffers at pH 5.5 (A and C) and 8.5 (B and D). All buffers were osmotically balanced using gluconate− as the substitute anion. Data shown represent the mean ± SEM (n ≥ 4). Where error bars cannot be seen, they lie within the symbol. Note the scale difference in the y axes.

References [+] :

Akyuz, Transport domain unlocking sets the uptake rate of an aspartate transporter. 2015, Pubmed