Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

Mukhtasimova, Nuriya; daCosta, Corrie J.B.; Sine, Steven M.

doi:10.1085/jgp.201611584

Cited by 48 publications

(89 citation statements)

References 50 publications

(68 reference statements)

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“…Dwell interval analysis of active periods demonstrated that the receptors have multiple components, indicating that each receptor oscillates between multiple functional states during receptor activation [8, 11, 13]. The pattern of dwell components also indicated that at ≥ 2 μM glutamate an optimal number of bound glutamate molecules achieves efficient receptor activation.…”

Section: Discussionmentioning

confidence: 99%

“…The mechanism of agonist activation has been studied extensively in vertebrate pLGIC members, such as the glycine (GlyR) [8], acetylcholine (AChR) [9–11], serotonin (5-HT 3 R) [12] and GABA A (GABA A R) [13–15] receptors, as well as ELIC, which is a bacterial pLGIC [16]. A detailed study of the biophysical properties of GluClR activation has not been undertaken, even though GluClRs constitute a major group of pLGICs, many organisms that express them are serious parasitic pests, or vectors for disease transmission and they are a major target for anthelminthic drugs.…”

Section: Introductionmentioning

confidence: 99%

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Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus

Atif¹,

Estrada-Mondragón²,

Nguyen³

et al. 2017

PLoS Pathog

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Ivermectin (IVM) is a widely-used anthelmintic that works by binding to and activating glutamate-gated chloride channel receptors (GluClRs) in nematodes. The resulting chloride flux inhibits the pharyngeal muscle cells and motor neurons of nematodes, causing death by paralysis or starvation. IVM resistance is an emerging problem in many pest species, necessitating the development of novel drugs. However, drug optimisation requires a quantitative understanding of GluClR activation and modulation mechanisms. Here we investigated the biophysical properties of homomeric α (avr-14b) GluClRs from the parasitic nematode, H. contortus, in the presence of glutamate and IVM. The receptor proved to be highly responsive to low nanomolar concentrations of both compounds. Analysis of single receptor activations demonstrated that the GluClR oscillates between multiple functional states upon the binding of either ligand. The G36’A mutation in the third transmembrane domain, which was previously thought to hinder access of IVM to its binding site, was found to decrease the duration of active periods and increase receptor desensitisation. On an ensemble macropatch level the mutation gave rise to enhanced current decay and desensitisation rates. Because these responses were common to both glutamate and IVM, and were observed under conditions where agonist binding sites were likely saturated, we infer that G36’A affects the intrinsic properties of the receptor with no specific effect on IVM binding mechanisms. These unexpected results provide new insights into the activation and modulatory mechanisms of the H. contortus GluClRs and provide a mechanistic framework upon which the actions of drugs can be reliably interpreted.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus

Atif¹,

Estrada-Mondragón²,

Nguyen³

et al. 2017

PLoS Pathog

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“…The presence of multiple agonist-bound closed states is a general feature of the pLGIC superfamily and there is substantial evidence that the closed intermediate states stabilized by agonist binding have increased affinity for the agonist 11–14 . Fitting models that include activation intermediates to GlyR, nAChR and 5-HT 3 R single channel data have led to the proposal that the limited efficacy of partial agonists is due to their reduced ability to change the conformation to a short-lived pre-open intermediate (flipped/primed), rather than the reduced ability to open the receptor once the intermediate is reached 12, 15, 16 .…”

Section: Introductionmentioning

confidence: 99%

Mechanism of gating and partial agonist action in the glycine receptor

Zhu

Lape

et al. 2019

Preprint

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21 22 channel states, thus explaining their lesser efficacy, yet also populate agonist-bound open and 32 desensitized states. Measurements within the neurotransmitter binding pocket, as a function of 33 bound agonist, provide a metric to correlate the extent of agonist-induced conformational changes 34 to open channel probability across the Cys-loop receptor family. 35 36 to change the conformation to a short-lived pre-open intermediate (flipped/primed), rather than 62the reduced ability to open the receptor once the intermediate is reached 12,15,16 . 63Here we investigate the structural basis of partial agonist activation in GlyR, a paradigm 64Cys-loop receptor, exploiting extensive functional characterization showing that taurine and 65 GABA act as partial agonists [17][18][19] . Previous GlyR structures have shed light on the mechanism of 66 action of full agonists and antagonists 20-23 , but structures of the receptor bound to partial agonists 67 are still absent. Furthermore, the previously solved GlyR structures are in detergent micelles and 68 lack the M3/M4 cytoplasmic loop, factors that likely underlie the anomalously high Po of the 69 receptor constructs and the physiologically 'too large' open state of the ion channel pore 24,25 . 70Here we isolated the full length GlyR with native lipids and determined high resolution structures 71 of the receptor bound to glycine, taurine or GABA. The receptors were imaged by cryo-electron 72 microscopy (cryo-EM) in MSP nanodiscs or after solubilization by styrene maleic acid (SMA) 73 copolymers 26 , showing by MD simulations that the SMA complex adopts a physiologically relevant 74 open state. Importantly, cryo-EM structures of GlyR bound to taurine or GABA reveal, for the first 75 time, agonist-bound closed states as well as open and desensitized states. 76 Glycine-bound states 77The wild-type GlyR is robustly activated by glycine and single-channel analysis of clusters 78 of receptor activity, excluding the long-lived desensitized states, reveal a maximum open 79 probability (PO) of 97% at saturating concentrations of glycine (Fig. 1a). Macroscopic current 80 recordings of GlyR activity elicited by rapid application of 10 mM glycine to outside out patches 81show that the current decays to ~ 57% of peak after 1s application because of desensitization 82 (Extended Data Fig. 1a). Taken together, single channel and macroscopic results suggest that, 83 under steady state conditions, the glycine-bound GlyR substantially populates both open and 84 desensitized states. Hence, we reconstituted detergent-purified GlyR into nanodiscs or we directly 85 page 5 of 43 extracted the receptor using SMA copolymers, followed by preparation of cryo-EM grids in the 86 presence of 10 mM glycine. 87The glycine-bound receptor in nanodiscs (GlyR-nanodisc-gly) yielded a reconstruction 88 with a single 3D class at 3.2 Å resolution (Fig. 1c, Extended Data Fig. 7, Supplementary Fig. 1, 89 Supplementary Table 1). The receptor features are well resolved and there is clear density for 90 lipi...

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“…A similar phi pattern has been observed in CFTR (Sorum et al, 2015). Structures of GLIC having an O-like ECD and a C-like TMD (Prevost et al, 2012; Sauguet et al, 2014) and an approximate microsecond shut component in high-resolution electrophysiology recordings (“flip” or “primed”; Lape et al, 2008; Mukhtasimova et al, 2016) are more-direct evidence for an intermediate state or states in pLGIC gating.…”

Section: Introductionmentioning

confidence: 99%

A mechanism for acetylcholine receptor gating based on structure, coupling, phi, and flip

Gupta

Chakraborty

Vij

et al. 2016

Journal of General Physiology

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Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

Cited by 48 publications

References 50 publications

Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus

Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus

Mechanism of gating and partial agonist action in the glycine receptor

A mechanism for acetylcholine receptor gating based on structure, coupling, phi, and flip

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