Energy Landscape of the Substrate Translocation Equilibrium of Plasma-Membrane Glutamate Transporters

Abstract

Glutamate transporters maintain a large glutamate concentration gradient across synaptic membranes and are, thus, critical for functioning of the excitatory synapse. Mammalian glutamate transporters concentrate glutamate inside cells through energetic coupling of glutamate flux to the transmembrane concentration gradient of Na. Structural models based on an archeal homologue, GltPh, suggest an elevator-like carrier mechanism. However, the energetic determinants of this carrier-based movement are not well understood. Although electrostatics play an important role in governing these energetics, their implication on transport dynamics has not been studied. Here, we combine a pre-steady-state kinetic analysis of the translocation equilibrium with electrostatic computations to gain insight into the energetics of the translocation process. Our results show the biphasic nature of translocation, consistent with the existence of an intermediate on the translocation pathway. In the absence of voltage, the equilibrium is shifted to the outward-facing configuration. Electrostatic computations confirm the intermediate state and show that the elevator-like movement is energetically feasible in the presence of bound Na ions, whereas a substrate-hopping model is energetically prohibitive. Our results highlight the critical contribution of charge compensation to transport and add to results from previous molecular dynamics simulations for improved understanding of the glutamate translocation process.

A K+/Na+ co-binding state: Simultaneous versus competitive binding of K+ and Na+ to glutamate transporters

“…Cell culture and transfection HEK293 cells (American Type Culture Collection, catalog number CRL 1573) were cultured as described previously (48).…”

“…Series resistance was not compensated because of the small whole-cell currents carried by EAAC1, EAAT1, and EAAT2. The composition of the solutions for measuring forward transport currents was 140 mM NaMes/KMes, 2 mM Mg(gluconate) 2 , 2 mM Ca(gluconate) 2 , 10 mM HEPES, 10 mM glutamate, pH 7.3 (extracellular), and 130 mM KMes, 2 mM Mg(gluconate) 2 , 5 mM EGTA, 10 mM HEPES, pH 7.3 (intracellular), as published previously (48). For anion current recordings, intracellular Mes Ϫ was replaced by SCN Ϫ .…”

The high‐energy transition state of the glutamate transporter homologue GltPh

“…In contrast, we know relatively little about the structures of high‐energy transition states (TSs) that are essential to understand the kinetic mechanisms, how the kinetics are affected by post‐translational modifications, or small‐molecule modulators (Kortagere, Fontana et al , 2013; Li, Hasenhuetl et al , 2015; Czuba, Hillgren et al , 2018) or how to develop drugs that modulate transporter activities (Rives, Javitch et al , 2017). While many computational approaches are used to map the energy landscapes of the transporters (Weng, Fan et al , 2010; Espinoza‐Fonseca & Thomas, 2011; Jiang, Shrivastava et al , 2011; Stolzenberg, Khelashvili et al , 2012; Weng, Fan et al , 2012; Gur, Zomot et al , 2013; Stelzl, Fowler et al , 2014; Gur, Zomot et al , 2015; Moradi, Enkavi et al , 2015; Liao, Marinelli et al , 2016; Cheng, Kaya et al , 2018; Selvam, Mittal et al , 2018; Wang, Albers et al , 2018), experimental data reporting on TSs are scarce (Leninger, Sae Her et al , 2019; Wu, Wynne et al , 2019).…”

Millisecond dynamics of an unlabeled amino acid transporter

“…3d). We term this state "intermediate state" in the remainder of the manuscript, but cannot determine whether this state corresponds to an intermediate state previously observed structurally or computationally 23,[44][45][46][47] . As expected, the state-assignment algorithm mainly assigned three states in traces with elevated SNR > 2.…”