Binding thermodynamics of a glutamate transporter homolog

Abstract

Glutamate transporters catalyze concentrative uptake of the neurotransmitter into glial cells and neurons. Their transport cycle involves binding and release of the substrate on the extra- and intracellular sides of the plasma membranes, and translocation of the substrate-binding site across the lipid bilayers. The energy of the ionic gradients, mainly sodium, fuels the cycle. Here, we used a cross-linking approach to trap a glutamate transporter homologue from Pyrococcus horikoshii in key conformational states with substrate-binding site facing either the extracellular or intracellular sides of the membrane to study their binding thermodynamics. We show that the chemical potential of sodium ions in solution is exclusively coupled to substrate binding and release, and not to substrate translocation. Despite the structural symmetry, the binding mechanisms are distinct on the opposite sides of the membrane and more complex than the current models suggest.

On the principle of ion selectivity in Na + /H + -coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

“…The cluster analysis was carried out using METAGUI (66,67) (51-53). Reassuringly, ITC studies of membrane proteins in which the binding stoichiometry could be actually determined, for example those of substrate binding to the Glt Ph symporter, confirmed the n value deduced from crystal structures (54).…”

Direct visualization of glutamate transporter elevator mechanism by high-speed AFM

“…Thus, we believe that the concentrations of Na + ions and Asp, as seen by the transporter, are the same on the opposite sides of the membrane. Glt Ph shows highly cooperative and tight binding of Na + ions and Asp from both the extracellular and intracellular sides of the membrane (21). Under our conditions, we expect the apparent dissociation constants for Na + and Asp to be below 10 mM and 1 nM, respectively.…”

Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog

“…We first set out to explore the effects of 3L4 cleavage on protein conformational sampling by using Hg 2ϩ -based crosslinking, which is a well established method used successfully with Glt Ph (8,9,22 is also difficult to quench the Hg 2ϩ cross-linking reaction with commonly used quenching compounds, such as N-ethylmaleimide or MMTS. In the absence of a quenching reagent, when attempting to cross-link cleaved Glt Ph in detergent solution, we observed protein bands on SDS-polyacrylamide gels corresponding to cross-link formation between intra-protein fragments and also, surprisingly, inter-subunit fragments.…”

“…For all pairs attempted, the introduced cysteine pairs either did not cross-link using MTS-1-MTS (however, one of these examples, L66C/S300C, does cross-link robustly with Hg 2ϩ ; its structure has been published recently (22)) or a disulfide would form during purification despite efforts to prevent it. These disulfide bonds could only be reduced under harsh conditions that would render the protein inactive.…”