Na ⁺ -dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters

Abstract

Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh. A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.

On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog

“…The order and timescale of these events is, as discussed, artefactual, but the resulting configuration, only occupied by Na + in Na1 and Na3, is a mechanistically relevant state in physiological conditions [34][35][36]. In agreement with recently-published structures of inward-facing Glt Tk [10] and outward-facing Glt Ph [38], we observe that this outward-facing, partiallyoccupied state preserves many of the key features of the holo-state structure [19]. For example, the ion-pair between Arg401 and Asp394, which primes Arg401 for substrate coordination (Figs.…”

“…Invariably, the absence of L-Asp and the third ion at the Na2 site leads to the opening of this hairpin towards the extracellular space, which after 1 µs of simulation adopts a conformation closely resembling that observed in structures of Glt Ph and Glt Tk bound to blockers [5,10] (Fig. 6g, h), and in a recently-reported structure of Glt Ph in the same partially occupied state [38]. By contrast, extended simulations of the fully-occupied transporter (made possible by the NBFIX correction described above) demonstrate that the presence of all ions and the substrate correlates with HP2 favoring the occluded conformation (Fig.…”

“…As mentioned, experimental mutation of the methionine in the NMDGT motif has clear effects on the function of both EAAT2/3 [21,23,33] and Glt Ph [37][38][39]. Some of these observations, taken together with the nature of the Na + -methionine interaction discussed above, could be interpreted as evidence that this methionine is essential for defining the 3:1 stoichiometry of the coupled ion-substrate transport reaction.…”

“…These results indicate that the energy landscape governing the dynamics of HP2, and by extension the occlusion of the transporter and the viability of the alternating-access transition, is indeed re-shaped by the occupancy or vacancy of the ion and substrate binding sites. In other words, opening of HP2 does appear to serve as the The protein backbone is shown as cartoon helices, and key side chains are shown as sticks structural mechanism that prevents uncoupled transport, as suggested elsewhere [37,38]. Further experimental and computational investigations will be required to continue to examine this hypothesis and fully clarify its molecular basis.…”

“…However, this cooperativity is greatly suppressed when Met314 (Met311 in Glt Ph ) is replaced by Ala or Leu [37], as is the substrate affinity. Although no structures have been reported for any of these mutants, Trp fluorescence measurements show that the pattern of conformational changes in HP2 in response to ion and substrate binding [19,37] is also subtly altered upon mutation of Met314 [38,39].…”