Hetero-oligomerization of Neuronal Glutamate Transporters

Doreen Nothmann,Ariane Leinenweber,Delany Torres-Salazar,Peter Kovermann,Jasmin Hotzy,Armanda Gameiro,Christof Grewer,Christoph Fahlke

doi:10.1074/jbc.m110.187492

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.

Highlights

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system
Blue native (BN)-PAGE of full lysates from cells expressing YFP-rEAAT4 resulted in two different fluorescent bands that correlate to non- or coreglycosylated and complex-glycosylated forms of homotrimers (Fig. 1B, lane 2) [14]
We here demonstrate that EAAT3 and EAAT4, but neither EAAT1 and EAAT2 nor EAAT2 and EAAT3, co-assemble into stable heterotrimers in heterologous expression systems

Summary

EXPERIMENTAL PROCEDURES

Heterologous Expression of EAATs—Coding regions of rat EAAT1, human EAAT2, rat and human EAAT3, rat EAAT4, and human SLC26A9 were subcloned into pcDNA3.1 or pRcCMV using PCR-based strategies. Current recordings from oocytes expressing hEAAT3 were usually performed 1 day after injection. Cells were only incorporated into the analysis if current amplitudes measured at different times with the same substrate concentration perfectly superimpose. Electrophysiological recordings were performed 24 h after the transfection for 3 days In these experiments, the extracellular solution contained 140 mM NaMES, 2 mM CaMES2, 2 mM MgMES2, and 30 mM HEPES (pH 7.4/NaOH). To estimate the concentration of photolytically released glutamate, a standard glutamate concentration of 100 ␮M was applied to the cells by rapid perfusion before and after photolysis experiments, and the steadystate current amplitude was used to calculate the free glutamate concentration from the dose-response curve [26]. Current responses to the laser pulse were low pass-filtered at 3 kHz (100-␮s time resolution) and recorded with a 100-kHz sampling rate. The so-obtained substrate dependences were normalized to the maximum current amplitude, fit with Hill equations,

Imaxsubstratensubstraten ϩ KDn ϩ

RESULTS

DISCUSSION