Individual subunits of the glutamate transporter EAAC1 homotrimer function independently of each other

Abstract

Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. Here, we have tested the effect of multimerization on the transporter function. To do so, we coexpressed EAAC1(WT) with the mutant transporter EAAC1(R446Q), which transports glutamine but not glutamate. Application of 50 microM glutamate or 50 microM glutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 and 130 pA, respectively. Application of both substrates at the same time generated an anion current of 297 pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purely additive. This result is unexpected for anion permeation through a central pore but could be explained by anion permeation through independently functioning subunits. To further test the subunit independence, we coexpressed EAAC1(WT) and EAAC1(H295K), a transporter with a 90-fold reduced glutamate affinity as compared to EAAC1(WT), and determined the glutamate concentration dependence of currents of the mixed transporter population. The data were consistent with two independent populations of transporters with apparent glutamate affinities similar to those of EAAC1(H295K) and EAAC1(WT), respectively. Finally, we coexpressed EAAC1(WT) with the pH-independent mutant transporter EAAC1(E373Q), showing two independent populations of transporters, one being pH-dependent and the other being pH-independent. In conclusion, we propose that EAAC1 assembles as trimers of identical subunits but that the individual subunits in the trimer function independently of each other.

Figure 1

(A) Topology of EAAC1 and the location of the mutated amino acid residues. (B) Graphical illustration of the transport modes studied. (C) Glutamate does not activate EAAC1_R446Q. Typical inward currents are shown in response to application of 500 μM alanine to a cell expressing EAAC1_R446Q (left panel). The middle panel shows the response of the same cell to application of 500 μM glutamate (140 mM NaSCN, 10 mM alanine, and 10 mM glutamate in the recording pipette solution). Application of 500 μM alanine in the presence of intracellular KSCN (forward transport conditions) evokes very little current (right panel). (D) Dose response relationships for alanine and glutamate under recording conditions as in (A). The solid lines represent best fits according to a Michaelis-Menten-like relationship. All recordings were performed at 0 mV transmembrane potential.

Figure 2

Anion current responses to glutamate and glutamine of WT/R446Q mixed transporter populations are purely additive. (A) 50 μM glutamate (left trace), 50 μM glutamine (right trace), and 50 μM glutamate + 50 μM glutamine (middle trace) were applied to a cell expressing both EAAC1_WT and EAAC1_R446Q after transfection with a 1:1 mixture of the respective cDNAs. Line A represents the current level evoked by glutamate application and B the current evoked by glutamine application. The baseline was adjusted to 0. Currents were recorded in the exchange mode (140 mM NaSCN, 5 mM glutamine and 5 mM glutamate in the recording pipette solution). (B) Statistical analysis of the data shown in (A) (n = 4). (C) Ratio of experimentally determined and expected currents as a function of the ratio of transfected cDNA concentration for purely statistical coassembly of the trimer. The expected currents were calculated from a binomial distribution with probabilities of finding one of the two subunits in the trimeric assembly of 0.2 (ratio 1:4), 0.5 (ratio 1:1), and 0.8 (ratio 4:1), respectively.

Figure 3

Thiol-specific crosslinking of the heterologously expressed wild type EAAC1 and the cysteine-less mutant of EAAC1. Hypotonically washed membranes derived from EAAC1_WT (A) or EAAC1_cysless (B) expressing HEK293 cells were incubated with 25 μM BMDB (A, B, lanes 2, 3, 4, 5, respectively). The crosslinking reaction was stopped after incubation times of 10, 30, 60, and 120 min. Controls (non-crosslinked membranes before and after the experimental procedure; starting material: lanes 1; post-experimental: lanes 6) and crosslinking products (10 μg total membrane protein per lane) were separated by 6% SDS-PAGE and were immunoblotted with EAAC1-specific antibodies. Molecular mass markers (left panels: MW) are indicated in kDa. The data shown are representative of at least three independent experiments.

Figure 4

Co-immunoprecipitation results. EAAC1_WT and EAAC1_R446Q were co-immunoprecipitated with a EAAC1_WT-YFP fusion protein by using an anti-GFP antibody. Total (T), unbound (U), washed (W) and eluted (E) samples were loaded on 10% SDS-PAGE. The corresponding amount of anti-GFP was also loaded as a control (C). Protein was transferred to a nylon membrane and detected by anti-EAAC1 antibody and ECL reagent. The left four lanes were samples from cells transfected with EAAC1_R446Q alone; the right four lanes were samples from cells transfected with EAAC1_R446Q plus EAAC1_WT-YFP.

Figure 5

Forward transport of EAAC1_WT is not inhibited in the presence of equal amounts of EAAC1_R446Q. (A) 50 μM glutamate (left panel), 200 μM alanine (middle panel), and 50 μM glutamate + 200 μM alanine (right panel) were applied to a cell expressing both EAAC1_WT and EAAC1_R446Q after transfection with a 1:1 mixture of the respective cDNAs. The line marked Glu represents the current level evoked by glutamate application, Ala the current evoked by alanine application, and Glu + Ala the current level in the presence of both substrates. The baseline was adjusted to 0. Anion currents were recorded in the forward transport mode in the presence of permeating anions (140 mM KSCN in the recording pipette solution). (B) Glutamate dose-response curves recorded in the absence (open symbols) and the presence (closed symbols) of 500 μM extracellular alanine. The data shown as the circles were recorded in the presence of intracellular KSCN (forward transport mode). The triangles represent experiments performed in the homoexchange mode (140 mM NaSCN, 5 mM glutamate, 5 mM alanine internal) in the presence of 500 μM extracellular alanine. The data were obtained at 0 mV transmembrane potential. The dashed line represents a fit to a Michaelis-Menten-like equation with an additional y-offset parameter. The solid and the dotted line are fits according to a Michaelis-Menten equation. (C) Transport currents induced in EAAC1_WT and EAAC1_R446Q coexpressing cells by application of 50 μM glutamate (left panel), 50 μM glutamine (middle panel), and 50 μM glutamate + 50 μM glutamine (right panel). The baseline was adjusted to 0. Currents were recorded in the forward transport mode in the absence of permeating anions (140 mM KCl in the recording pipette solution). (D) Voltage dependence of transport currents under conditions similar as in (C). Transport currents were recorded in the presence of 50 μM glutamate (closed circles), 50 μM glutamine (open circles), and 50 μM glutamate + 50 μM glutamine (triangles).

Figure 6

Inhibition of individual transporter subunits by competitive inhibitors does not exert a dominant negative effect. (A) Left panel: Anion current evoked by the application of 500 μM alanine to a mixed population of EAAC1_WT and EAAC1_R446Q. Right panel: Application of 100 μM TBOA, a competitive inhibitor of EAAC1_WT, to the same cell evokes an outward current (upper trace). The lower trace shows the response to both 500 μM alanine and 100 μM TBOA. All currents were recorded in the homoexchange mode. (B) Left panel: Anion current evoked by the application of 50 μM glutamate to a mixed population of EAAC1_WT and EAAC1_R446Q. Right panel: Application of 1 mM Bzl-Ser, a competitive inhibitor of EAAC1_R446Q, to the same cell evokes an outward current (upper trace). The lower trace shows the response to both 50 μM glutamate and 1 mM Bzl-Ser. The arrow represents the total glutamate-induced current response in the absence and presence of Bzl-Ser. All currents were recorded at 0 mV voltage with a SCN⁻ containing pipette solution. (C) Statistical analysis of the data shown in A and B (n = 4).

Figure 7

(A) Dose response relationship of a mixed population of EAAC1_WT and EAAC1_H295K transporters activated by glutamate. The dashed lines represent the dose response relationships of pure EAAC1_WT and EAAC1_H295K transporters. The solid line represents the best fit of a sum of two Michaelis-Menten-like relationships to the data, assuming an independent population of WT and mutant transporters. The dotted line represents a Michaelis-Menten-like relationship calculated with a K_m intermediate between wild-type and mutant EAAC1. The cDNAs for the mutant and wild-type transporters were mixed in a 1:1 ratio. The recording pipet contained 140 mM NaSCN and 10 mM glutamate (V_m = 0 mV). (B) Current responses to 1 mM glutamate application to HEK293 cells expressing wild-type EAAC1 (left panel), EAAC1_H295K (right panel), and a mixed population of wild-type transporter and EAAC1_H295K under the same conditions as in (A).

Figure 8

Dose response relationship of a mixed population of EAAC1_WT and EAAC1_E373Q transporters activated by glutamate at pH 7.4 (open circles) and pH 10.0 (closed circles). The dashed lines represent the dose response relationships of EAAC1_WT at pH values of 7.4 and 10.0. The solid lines represent the best fit of a sum of two Michaelis-Menten-like relationships to the data at pH 7.4 and 10.0, assuming an independent population of WT and mutant transporters (ratio of cDNA concentrations of 1:1). The data were collected in the presence of 140 mM Na⁺ and 10 mM glutamate in the cytosol (homoexchange conditions).

Figure 9

(A) Proposed model for the independent action of subunits of EAAC1_WT (dark grey, transports Glu) and EAAC1_R446Q (light grey, transports either Ala or Gln) in the heterotrimeric unit. The model shows a side-view of the transporter according to the crystal structure of Yernool et al. (12). The third subunit is represented by the dashed line. The cylinder represents the anion permeation pathway. (B and C) Comparison of experimental currents (B) and expected currents (C) induced upon coapplication of glutamate and glutamine to the mixed population of transporters according to three different models. In the left model both subunits work independently and anion permeation is through pathways on individual subunits (see also (A)). In the middle model anion permeation is through a central pathway and needs binding of both ligands to be activated. In the right model anion permeation is through a central pathway and needs binding of only one of the two ligands to be activated. The expected currents were calculated based on a binomial distribution with a probability of finding each of the two subunits in a trimeric assembly of 0.5 (1 : 1 coexpression of wild-type and mutant transporter).