Hetero-oligomerization of Neuronal Glutamate Transporters

Abstract

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo-and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.

The importance of the excitatory amino acid transporter 3 (EAAT3)

“…The protein was crystalized as a homo-trimer with each subunit functioning independently, which was also confirmed for the mammalian orthologs (Haugeto et al, 1996, Gendreau et al, 2004, Grewer et al, 2005). Interestingly, there is evidence that hetero-trimeric quaternary structures are possible, as seen with recombinant EAAT3 and EAAT4 expressed in tsA201 cells (Nothmann et al, 2011). …”

Human CLC-K Channels Require Palmitoylation of Their Accessory Subunit Barttin to Be Functional

“…For analysis of cell surface expression of CLC-K/barttin channels, MDCK II cells were incubated for 40 min with 0.25 mg of biotin (EZ link sulfo-NHS-SS-biotin; Pierce) before cell lysis (28,37). Biotinylated proteins were purified using NeutrAvidin affinity chromatography (Pierce) and separated by electrophoresis on 12% SDS-polyacrylamide gels.…”

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

“…Surface Protein Biotinylation-Cell surface expression of hClC-Kb channels was investigated using a modification of cell surface biotinylation methods described previously (26,35). MDCK II cells (10-cm dishes) were grown to confluency after transfection, washed with PBS, and incubated with 1 mg of EZ linked NHS-Sulfo-SS-biotin (Thermo Scentific) in PBS for 30 min.…”