Elucidating the Anion Channel Gating Mechanism in Excitatory Amino Acid Transporters

Abstract

In the mammalian central nervous system, excitatory amino acid transporters (EAATs) are responsible for the clearance of glutamate after synaptic release. They complete this task through a secondary active transport mechanism, which is critical to maintain extracellular glutamate under neurotoxic levels. In addition, EAATs operate as substrate-gated anion channels, function that has been proposed to influence cellular excitability. Although these two mechanisms are known to be thermodynamically uncoupled, there is evidence of conformational changes involved in anion channel gating being tightly coupled to transitions within the transport cycle. We have recently identified a conserved positively charged residue (R388, hEAAT1) in transmembrane domain 7 (TM7), which seems to be a crucial player for such a coupling. Substituting R388 with a negatively charged amino acid drives the protein into a constitutive open channel state, while at the same time limits the transport cycle. We here combine electrophysiological recordings and molecular dynamic simulations aiming to better understand the mechanism that controls the switch between transport mode and channel mode. As a first step, 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 and the hairpin loop 1 (HP1) which may be coordinating the anion channel gating mechanism. Moreover, our preliminary results show that mutating E377 (HP1) to a positively charged residue, similar to R388D/E, yields a large anion conductance and a significantly reduced transport activity (less than 1%). Our data provide additional insights into the mechanism by which substrate gate the EAAT-associated anion conductance and suggest that intramolecular interactions between R388 and nearby residues are a crucial piece of the gating mechanism in EAAT-associated anion channels.