Chloride-dependent conformational changes in the GlyT1 glycine transporter

Abstract

The human GlyT1 glycine transporter requires chloride for its function. However, the mechanism by which Cl- exerts its influence is unknown. To examine the role that Cl- plays in the transport cycle, we measured the effect of Cl- on both glycine binding and conformational changes. The ability of glycine to displace the high-affinity radioligand [3H]CHIBA-3007 required Na+ and was potentiated over 1000-fold by Cl-. We generated GlyT1b mutants containing reactive cysteine residues in either the extracellular or cytoplasmic permeation pathways and measured changes in the reactivity of those cysteine residues as indicators of conformational changes in response to ions and substrate. Na+ increased accessibility in the extracellular pathway and decreased it in the cytoplasmic pathway, consistent with stabilizing an outward-open conformation as observed in other members of this transporter family. In the presence of Na+, both glycine and Cl- independently shifted the conformation of GlyT1b toward an outward-closed conformation. Together, Na+, glycine and Cl- stabilize an inward-open conformation of GlyT1b. Our results suggest that Cl- acts by interacting with a conserved glutamine to allow formation of an ion pair that stabilizes the closed state of the extracellular pathway. Molecular dynamics simulations of a GlyT1 homologue suggest that this ion pair is formed more frequently as that pathway closes. Mutation of the glutamine blocks the effect of Cl-, and substituting it with residues of opposite charge lead to inverse conformations of the transporter. These results provide novel and unexpected insight into the role of Cl- in this family of transporters.

Structure-based Discovery of Conformationally Selective Inhibitors of the Serotonin Transporter

“…Because Na + and Cl - influence the conformation of SERT and related transporters (Zhang et al ., 2021; Zhang et al ., 2016), we tested the effect of ‘8090 and ‘8219 on SERT conformation, seeking to understand if the new inhibitors bound to the inward-open conformation, as targeted, and akin to ibogaine, the outward open conformation, akin to cocaine and SSRIs, or an intermediate state. We used SERT mutants, depleted in reactive endogenous cysteine residues, and containing novel cysteine residues in either cytoplasmic or extracellular pathways, as previously used to determine the conformational effects of ibogaine on SERT (Jacobs et al, 2007b) (see Figure S6).…”

“…The conformations revealed by X-ray and cryo-EM studies correlate with functional studies of NSS transporters using single molecule FRET, electron spin resonance and cysteine accessibility that demonstrate the extracellular pathway becoming more accessible in the presence of Na + as the cytoplasmic pathway closes (Claxton et al, 2010; Fenollar-Ferrer et al, 2014; Forrest et al ., 2008; Quick et al, 2006; Tavoulari et al, 2016; Zhang et al, 2021; Zhao et al, 2010). Addition of substrate, and of Cl - in the case of mammalian neurotransmitter transporters, closes the extracellular pathway and opens the cytoplasmic pathway (Kazmier et al, 2014; Quick et al ., 2006; Zhang et al ., 2021; Zhang et al, 2018; Zhang et al, 2016; Zhao et al, 2011). SERT’s conformation also responds to the binding of antidepressants and of other drugs, with antidepressants and cocaine stabilizing the outward-open conformation while ibogaine and noribogaine stabilize the inward-open conformation (Coleman et al ., 2019; Forrest et al ., 2008; Jacobs et al ., 2007a; Tavoulari et al, 2009).…”

Reconstitution of GABA, Glycine and Glutamate Transporters

“…Proteoliposomes were loaded with various internal media: 400 mM K + (400 mM potassium ions, 250 mM HEPES, 100 mM citric acid, and 1% v/v glycerol), 200 internal proteoliposome pH in reconstituted transporters, which was expected to neutralize the introduced negative charge, increased the rate of transport of the mutant, but not of the wild-type, by an order of magnitude. These experiments, performed on proteoliposomes, provided a fundamental piece of evidence for the location of the chloride site in GAT1 [214] at an equivalent position as was located in SERT [215] and GlyTs [216].…”

Pre-steady-state Kinetic Analysis of Amino Acid Transporter SLC6A14 Reveals Rapid Turnover Rate and Substrate Translocation

“…Other members of the SLC6 family, including the GlyTs, are chloride dependent ( Zafra and Gimenez, 1986 ; Aragon et al, 1987 ; Liu et al, 1993 ; Zafra et al, 1997 ; Lopez-Corcuera et al, 1998 ; Roux and Supplisson, 2000 ; Zhang et al, 2021 ). Consistently, SLC6A14 was also be found to be chloride dependent ( Sloan and Mager, 1999 ; Nakanishi et al, 2001 ; Hatanaka et al, 2004 ; Umapathy et al, 2004 ; Karunakaran et al, 2008 ).…”