Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance

Abstract

Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-D-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate flux and two-electrode voltage clamp recording. EAAT5-mediated L-glutamate uptake is sodium- and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium- and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.

Figure 1

EAAT5 is a member of the human glutamate transporter gene family. (A) The predicted amino acid sequences of human EAAT5 is shown in alignment with the other known human EAAT subtypes (1, 2). In this alignment, amino acid residues identical in four of the five sequences are shown in white on black lettering to illustrate the extensive amino acid sequence conservation in this gene family. Although the transmembrane topology of these transporters is not well defined, one possible model is indicated here. The poorly conserved amino- and carboxy-terminal sequences are likely to be intracellular, and eight regions with strong transmembrane (TM) scores are indicated by bars and suggested orientation (i, inside; o, outside). The large conserved hydrophobic sequence (LCHS) indicated between TMs VII and VIII may be membrane-associated. Possible N-linked glycosylation sites (N-X-S or T) in a large extracellular loop are boxed. (B) The carboxyl terminus of EAAT5 conforms to a sequence motif (E-S or T-X-V) involved in subcellular targeting. NMDA receptor subunits NR2A and NR2B (12) and potassium channel Kv1.4 (13) interact with PSD-95 via their C termini.

Figure 2

EAAT5 mRNA is most abundantly expressed in the retina. The EAAT5 cDNA was used for hybridization of Northern-blotted poly(A)⁺ RNAs (2 μg) from retina and other human tissues. The retinal signal, a single 3.1-kb band, was overexposed here so that additional EAAT5-hybridizing bands in other tissue RNAs could be seen. Lower, the same blots were hybridized with a β-actin probe to control for RNA loading in each lane.

Figure 3

Pharmacology of EAAT5-mediated uptake and currents. (A) Uptake of [³H]-l-glutamate (100 μM) in oocytes voltage-clamped at −60 mV in normal Ringers solution (96 mM NaCl) (normal) or sodium-free (0 Na⁺) or chloride-free Ringers (0 Cl⁻) or voltage-clamped at +10 mV in normal Ringers. Uptake was sodium- and voltage-dependent. Additionally, uptake (−60 mV, normal Ringers) was blocked by coapplication of 100 μM THA or tPDC. ∗, Differs significantly from uptake measured in control uninjected oocytes. Data are the average of five cells each. (B) Dose- and voltage-dependent steady-state currents due to the application of l-glutamate to EAAT5-expressing oocytes. Concentration of l-glutamate indicated in the legend. Data are the average of seven cells. (C) Steady-state current elicited by 100 μM l-glutamate (squares) is blocked by coapplication of 100 μM tPDC (triangles); 100 μM tPDC alone (circles) elicits a small outward current at hyperpolarized potentials. Data are the average of four cells. (D) Steady-state current elicited by 100 μM l-glutamate (squares) is blocked by coapplication of 100 μM THA (triangles); 100 μM THA alone (circles) elicits a small outward current at hyperpolarized potentials. Data are the average of six cells. All error bars represent SEM. Error bars smaller than the symbols are not shown.

Figure 4

Ionic dependence of EAAT5-mediated currents. (A) Steady-state current elicited by 100 μM l-glutamate (circles) is abolished when external sodium is replaced with NMDG (squares). Data are the average of five cells. (B) Steady-state current elicited by 100 μM l-glutamate in normal chloride (circles) or when external chloride was replaced with gluconate (squares). Removal of external chloride ions blocks outward current at positive potentials and has no effect at negative potentials. Data are the average of five cells. (C) Steady-state current elicited by 100 μM l-glutamate in normal oocytes (circles) or in oocytes dialyzed in chloride-free solution for >48 h (squares). No currents are seen in oocytes dialyzed to remove chloride ions. Data are the average of 10 cells for each condition. (D) Steady-state current elicited by 100 μM l-glutamate in normal chloride (circles) or when most external chloride was replaced with nitrate (squares). l-glutamate elicited current in nitrate produces a large outward current. Data are the average of six cells. All error bars represent SEM. Error bars smaller than the symbols are not shown.