Intermediate closed state for glycine receptor function revealed by cysteine cross-linking

Abstract

Pentameric ligand-gated ion channels (pLGICs) mediate signal transmission by coupling the binding of extracellular ligands to the opening of their ion channel. Agonist binding elicits activation and desensitization of pLGICs, through several conformational states, that are, thus far, incompletely characterized at the structural level. We previously reported for GLIC, a prokaryotic pLGIC, that cross-linking of a pair of cysteines at both sides of the extracellular and transmembrane domain interface stabilizes a locally closed (LC) X-ray structure. Here, we introduced the homologous pair of cysteines on the human α1 glycine receptor. We show by electrophysiology that cysteine cross-linking produces a gain-of-function phenotype characterized by concomitant constitutive openings, increased agonist potency, and equalization of efficacies of full and partial agonists. However, it also produces a reduction of maximal currents at saturating agonist concentrations without change of the unitary channel conductance, an effect reversed by the positive allosteric modulator propofol. The cross-linking thus favors a unique closed state distinct from the resting and longest-lived desensitized states. Fitting the data according to a three-state allosteric model suggests that it could correspond to a LC conformation. Its plausible assignment to a gating intermediate or a fast-desensitized state is discussed. Overall, our data show that relative movement of two loops at the extracellular-transmembrane interface accompanies orthosteric agonist-mediated gating.protein conformation | cys-loop receptor | signal transduction P entameric ligand-gated ion channels (pLGICs) are major brain receptors mediating signal transduction (1). They transduce agonist binding within their extracellular domain (ECD) into the opening of their ion channel within their transmembrane domain (TMD). This activation process, occurring in the millisecond time range, is followed by slower current decay, corresponding to desensitization processes of channel closing on prolonged agonist application. These events involve the major resting, active, and desensitized states, but also several intermediate states, which correspond to local energy minima within the conformational pathways of the allosteric transitions and are significantly visited during the conformational reorganizations.In the early 1980s, electrophysiological and rapid mixing binding experiments on nicotinic acetylcholine receptors (nAChRs) demonstrated that desensitization is at least biphasic, with a 10-to 100-ms fast desensitization followed by a 1-to 60-s slow desensitization process (2, 3). Time-resolved affinity labeling showed that the fast-and slow-desensitized states are structurally distinct (4), and electrophysiological recordings suggest that the multiexponential desensitization processes apply to other pLGICs (5). Overall, those data were represented by a minimal four-state (at least) allosteric model in which a central intermediate state accounts for fast desensitization (2, 6-8). In a...

Allosteric modulation as a unifying mechanism for receptor function and regulation

“…Similarly, the recently identified structure of the eukaryotic GABA A β 2 homopentamer is consistent with a D state. A “locally closed” state of GLIC exhibits the conformation of the active state, but with a closed channel and plausibly corresponds to an I, fast‐desensitised or intermediate state. Thus, with an increase in structural models for discrete conformational channel states, a mechanistic understanding of the transitions between them is now becoming accessible.…”

Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors

“…Parallel evidence of ECD-TMD coupling is found in GLIC mutants with an engineered disulfide cross-link between extracellular loop 2 and the M2-M3 linker, leading to a “locally closed” configuration (Prevost et al, 2012). Gating defective mutant Gly receptors with “locally closed” disulfide cross-links retain gating potentiation by propofol, suggesting that the ECD-TMD interface does not mediate this anesthetic effect (Prevost et al, 2013). Instead, based on the evidence presented above (section 4.…”

Protein dynamics and the allosteric transitions of pentameric receptor channels

“…Finally, several fast and slow desensitized I and D states and uncoupled forms of prokaryotic receptors have been identified, and their structures are currently under investigation. For example, the locally closed state (Prevost et al 2012 ) might be a fast desensitized I state (Prevost et al 2013 ), and the closed state of ELIC—often considered to be a resting state (see Calimet et al 2013 )—might represent a slowly desensitized D one (Velisetty et al 2012 ; Sauguet et al 2014a ). The ELIC structure has also been suggested to be in a “refractory” (Gonzalez-Gutierrez et al 2012 )/“uncoupled” state (daCosta and Baenziger 2013 ) (see Cecchini and Changeux 2014 for discussion).…”