Transport and channel functions in EAATs: the missing link

Abstract

Excitatory Amino Acid Transporters (EAATs) are integral membrane proteins that subserve multiple functions during neurotransmission. They are secondary-active transporters that catalyze the movement of glutamate molecules along with co-transported ions across the plasma membrane of neurons and glial cells. This function is critical to maintain glutamate homeostasis, to limit the diffusion of glutamate released at synapses, and to prevent excessive increases in extracellular glutamate, which has been associated with several neurodegenerative disorders. In addition, EAATs mediate a sodiumand substratedependent anion selective conductance. This channel activity allows the transporter to serve as a glutamate sensor that regulates cell excitability and may also promote electrogenic glutamate transport by clamping the cell at negative potentials. How these 2 functions exist and communicate within the homotrimeric glutamate transporter structure remains an unanswered question. Substrate transport and anion permeation in EAATs are widely accepted to be thermodynamically uncoupled. However, the requirement for glutamate and/or sodium to activate the channel suggests that transitions involved in anion channel opening and closing (“gating”) may be structurally coupled to conformational changes involved in substrate transport. Although this idea has been entertained by several groups, little direct evidence has been provided for how the 2 processes might be linked. In a recent study published in the Journal of Biological Chemistry, we identified a point mutation that drives the carrier in a substrateand voltage-independent constitutive open channel state, and displays a substantially reduced substrate transport activity. In this mutant, substitution of a highly conserved arginine (Arg-388 in EAAT1) with a negatively charged residue decreased substrate transport to 5% of wild type, whereas the macroscopic anion current amplitude was increased six-fold. In contrast to wild-type carriers, this large anion conductance measured in cells expressing R388D/E persisted at its maximum activity in the absence of sodium and glutamate, as well as throughout the entire voltage range from ¡100 to C60 mV. These observations indicate that the mutants exist in a constitutive open channel state and suggest that some of the conformational changes required for substrate transport are tightly coupled to anion channel gating. Recently, several groups in the field have begun to examine the nature of the conformational states of the protein that facilitate anion permeation. Crystallographic data from Verdon and Boudker captured a stable intermediate conformation of at least one of the protomers, which consists of a small (~3.5 A ) inward movement of the transport domain and was termed an intermediate outward facing state (iOFS). Because in this conformation the authors observed a cavity lined by conserved hydrophobic residues, they hypothesized it may provide the anion permeation pathway. Building on this observation, Shabaneh and colleagues performed cross-linking experiments of introduced cysteine pairs. They selected cysteine pairs that prevent the full transition to inward facing states but still allow conformational shifts between the outward and iOFS. The authors showed that cross-linking of these cysteines eliminated substrate transport while favoring