Individual Subunits of the Glutamate Transporter EAAC1 Homotrimer Function Independently of Each Other

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• 作者：Christof Grewer ; Poonam Balani ; Christian Weidenfeller ; Thorsten Bartusel ; Zhen Tao ; and ; and Thomas Rauen
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：September 6, 2005
• 年：2005
• 卷：44
• 期：35
• 页码：11913 - 11923
• 全文大小：671K
• 年卷期：v.44,no.35(September 6, 2005)
• ISSN：1520-4995

文摘

Glutamate transporters are thought to be assembled as trimers of identical subunits that linea central hole, possibly the permeation pathway for anions. Here, we have tested the effect ofmultimerization on the transporter function. To do so, we coexpressed EAAC1_WT with the mutant transporterEAAC1_R446Q, which transports glutamine but not glutamate. Application of 50 s/entities/mgr.gif">M glutamate or 50 s/entities/mgr.gif">Mglutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 and130 pA, respectively. Application of both substrates at the same time generated an anion current of 297pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purelyadditive. This result is unexpected for anion permeation through a central pore but could be explained byanion 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 ascompared to EAAC1_WT, and determined the glutamate concentration dependence of currents of the mixedtransporter population. The data were consistent with two independent populations of transporters withapparent glutamate affinities similar to those of EAAC1_H295K and EAAC1_WT, respectively. Finally, wecoexpressed EAAC1_WT with the pH-independent mutant transporter EAAC1_E373Q, showing two independentpopulations 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 thetrimer function independently of each other.