Individual subunits of the glutamate transporter EAAC1 homotrimer function independently of each other.
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.
Grewer
Christof
C
University of Miami School of Medicine, 1600 NW 10th Avenue, Miami, Florida 33136, USA. cgrewer@med.miami.edu
Balani
Poonam
P
Weidenfeller
Christian
C
Bartusel
Thorsten
T
Tao
Zhen
Z
Rauen
Thomas
T
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D050580:Excitatory Amino Acid Transporter 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats
D050580:Excitatory Amino Acid Transporter 3; D021122:Protein Subunits; C493398:Slc1a1 protein, rat; C120673:benzyloxyaspartate; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D000409:Alanine