Abstract
Excitatory amino acid transporters (EAATs) are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na+ and substrate binding. The activation of the anion channel by binding of Na+ alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3) and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na+-dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or “leak” channel activity. Choline or N-methyl-D-glucamine replacement of external Na+ ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na+ with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na+-independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system.
Highlights
Excitatory amino acid transporters (EAATs) are a family of five carriers expressed in the central nervous system (Danbolt, 2001)
The anion channel of EAAT4 is activated by binding of Na+, and open probability is further increased upon subsequent binding of glutamate, a process that is Na+-dependent (Fairman et al, 1995; Melzer et al, 2003; Kovermann et al, 2010)
We examined the effect of Na+ substitution with NMDG+ on radiolabeled substrate transport. 3,43H-L-glutamate uptake was significantly reduced in all isoforms (EAAT1: 92.2% ± 0.9%; EAAT2: 94.6% ± 2.0%; EAAT3: 94.6% ± 0.4%; and EAAT4: 103% ± 14.9% reduction; Figure 1C; p < 0.001 for all conditions)
Summary
Excitatory amino acid transporters (EAATs) are a family of five carriers expressed in the central nervous system (Danbolt, 2001). All members of the EAAT family function as secondary active transporters mediating the translocation of 1 L-glutamate molecule coupled to the co-transport of 3 Na+, 1 H+, and the counter-transport of 1 K+ ion (Zerangue and Kavanaugh, 1996; Watzke et al, 2000). This coupling allows the net inward movement of two positive charges with each glutamate translocated into the cytoplasm. Additional data have substantiated that EAAT5 is a ligand gated ionotropic receptor (Arriza et al, 1997; Wersinger et al, 2006; Gameiro et al, 2011) and support the need to investigate the roles of these anion channels in other EAAT isoforms
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