Intersubunit Interactions in EAAT4 Glutamate Transporters

Abstract

Excitatory amino acid transporters (EAATs) play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian CNS. A functional transporter is assembled as oligomer consisting of three subunits, each of which appears to transport glutamate independently from the neighboring subunits. EAATs do not only sustain a secondary-active glutamate transport but also function as anion channel. We here address the question whether intersubunit interactions play a role in poremediated anion conduction. We expressed a neuronal isoform, EAAT4, heterologously in Xenopus oocytes and mammalian cells and measured glutamate flux and anion currents under various concentrations of Na ϩ and glutamate. EAAT4 anion channels are active in the absence of both substrates, and increasing concentrations activate EAAT4 anion currents with a sigmoidal concentration dependence. Because only one glutamate molecule is cotransported per uptake cycle, the cooperativity between glutamate binding sites most likely arises from an interaction between different carrier domains. This interaction is modified by two point mutations close to the putative glutamate binding site, G464S and Q467S. Both mutations alter the dissociation constants and Hill coefficient of the substrate dependence of anion currents, leaving the concentration dependence of glutamate uptake unaffected. Our results demonstrate that glutamate carriers cooperatively interact during anion channel activation.

The Glutamate-Activated Anion Conductance in Excitatory Amino Acid Transporters Is Gated Independently by the Individual Subunits

“…EAAT4 has one of the highest chloride permeabilities among all the glutamate transporter subtypes (Fairman et al, 1995), and a recent study has suggested that the individual subunits of EAAT4 interact cooperatively in gating glutamate-activated anion currents (Torres-Salazar and Fahlke, 2006). We therefore measured FLIC in EAAT4 to determine whether or not its subunits interact cooperatively in gating the anion conductance.…”

“…In contrast, a recent study suggested that the individual subunits of EAAT4 function cooperatively in gating the anion conductance. The authors reported a sigmoidal concentration dependence for glutamate with Hill coefficients Ն3 and suggested that multiple glutamate binding sites must be occupied to activate the anion conductance (Torres-Salazar and Fahlke, 2006).…”

Hetero-oligomerization of Neuronal Glutamate Transporters

“…Electrophysiology-For expression in oocytes, cRNA was synthesized from MluI-linearized pTLN2-hEAAT3 (24) or from NheI-linearized pGEMHE-R501C rEAAT4 (20) templates through use of MESSAGE machine kits (Ambion, Austin, TX). Injection and handling of oocytes were performed as described elsewhere (25).…”

“…In contrast R501C rEAAT4, like WT rEAAT4, is inactivated by positive voltages (Fig. 2B) (8,20,25), resulting in decreased channel open probabilities and time-dependent current amplitude reductions (arrow) (8,25,34). WT hEAAT3 currents are greatly increased in the presence of extracellular glutamate, but are not responsive to serine (Fig.…”

“…hEAAT3/rEAAT4 heterotrimers therefore provide an excellent possibility to study potential intersubunit interactions. To separate currents conducted by hEAAT3 or rEAAT4 subunits, we mutated rEAAT4 at position 501 (R501C) to modify the substrate binding pocket in a way that glutamate cannot bind anymore, but serine or cysteine can bind instead (17,20,31). Fig.…”