Insights into the Gating Mechanism of Excitatory Amino Acid Transporters-Associated Anion Channel

Abstract

Excitatory Amino Acid Transporters (EAATs) control excitatory synaptic transmission in the brain. They complete this task through a secondary active transport mechanism. In addition, EAATs operate as substrate-gated anion channels. These two mechanisms are known to be thermodynamically uncoupled. However, emerging evidence demonstrates a tight structural coupling between them. We have recently identified a conserved positively charged residue (R388, hEAAT1) in transmembrane domain 7 (TM7), which is crucial for such a coupling. Substituting R388 with a negatively charged amino acid drives the carrier into a constitutive open channel state, while it limits the transport cycle. We here combine electrophysiological recordings and molecular dynamic simulations to characterize the mechanism that controls the switch between transport mode and channel mode. We have built a homology model of the mammalian isoform EAAT1 using the crystal structure of the archaeal orthologue GltPh as a template. Then using atomistic molecular simulations, we have identified residues in TM2, TM5, TM7 and the hairpin loop 1 (HP1) which may be coordinating with R388, the anion channel opening. Our results show that mutating E377 (HP1) to a positively charged residue, yields a large anion conductance and a significantly reduced transport activity (less than 1%), similar to R388D/E. These results suggest that an interaction E377-R388 is necessary to maintain an efficient transporter, and that affecting this interaction favors the equilibrium into an open channel and out of the transport cycle. Interestingly, a double mutant swapping these charges (R388D-E377R) virtually recovered the wild type phenotype, corroborating this hypothesis. Our data provide additional insights into the mechanism that facilitates opening and closing of the EAAT-associated anion channel and suggest that intramolecular interactions between R388 and nearby residues are a crucial piece of this process.