Structural interplay of anesthetics and paralytics on muscle nicotinic receptors

Abstract

General anesthetics and neuromuscular blockers are used together during surgery to stabilize patients in an unconscious state. Anesthetics act mainly by potentiating inhibitory ion channels and inhibiting excitatory ion channels, with the net effect of dampening nervous system excitability. Neuromuscular blockers act by antagonizing nicotinic acetylcholine receptors at the motor endplate; these excitatory ligand-gated ion channels are also inhibited by general anesthetics. The mechanisms by which anesthetics and neuromuscular blockers inhibit nicotinic receptors are poorly understood but underlie safe and effective surgeries. Here we took a direct structural approach to define how a commonly used anesthetic and two neuromuscular blockers act on a muscle-type nicotinic receptor. We discover that the intravenous anesthetic etomidate binds at an intrasubunit site in the transmembrane domain and stabilizes a non-conducting, desensitized-like state of the channel. The depolarizing neuromuscular blocker succinylcholine also stabilizes a desensitized channel but does so through binding to the classical neurotransmitter site. Rocuronium binds in this same neurotransmitter site but locks the receptor in a resting, non-conducting state. Together, this study reveals a structural mechanism for how general anesthetics work on excitatory nicotinic receptors and further rationalizes clinical observations in how general anesthetics and neuromuscular blockers interact.

Fig. 1. Etomidate stabilizes a desensitized state through TMD binding.

a Simplified gating cycle cartoon for the nicotinic receptor. Acetylcholine (ACh) binding shifts the conformational equilibrium toward a conducting, activated state, then to the more stable nonconducting desensitized state. b Two-electrode voltage clamp (TEVC) electrophysiology shows activation by acetylcholine and dose-dependent receptor antagonism by etomidate (Eto). R, A, and D indicate channel resting state, and activation and presumed desensitization components of current response. Red triangles highlight etomidate increasing apparent desensitization rate. Incomplete recovery in final acetylcholine application is from slow washout of etomidate, consistent with its membrane partitioning. Similar responses were seen from n = 8 independent cells. c Side view of the overall cryo-EM map of the receptor-etomidate-choline complex; α subunit is in green, β subunit in khaki, γ subunit in blue, δ subunit in violet, choline in red, etomidate in gold, cholesterols in tomato red, and phospholipids in pink; the lipid nanodisc is shown as a semitransparent surface. α_γ and α_δ subunits are named based on their neighboring complementary subunits. d Side view of two α subunits showing permeation pathway as dots representing solvent-accessible surface colored by diameter; purple is 2.8–5.6 Å diameter, while diameter > 5.6 Å is shown in blue. Thr2ʹ that forms the pore constriction is shown as sticks. e Etomidate-receptor interactions in α_γ subunit are shown as sticks with corresponding density. f Choline at α/γ interface.

Fig. 2. Succinylcholine inhibition and binding mechanism.

a TEVC electrophysiology illustrates lower efficacy of succinylcholine (SCC) compared to acetylcholine (ACh). Similar responses were seen in n = 8 independent cells. b Side view of succinylcholine-bound structure. Subunits are colored as in Fig. 1. Succinylcholine is shown as yellow spheres. c Side view of two α subunits showing permeation pathway as dots representing solvent-accessible surface colored by diameter; Thr2ʹ that forms pore constriction is shown as sticks. d Succinylcholine at α/γ interface. e Succinylcholine at α/δ interface.

Fig. 3. Rocuronium inhibition and binding mechanism.

a TEVC recording illustrating inhibition of acetylcholine evoked currents by rocuronium (ROC, chemical structure). Second recording shows effect of rocuronium pre-application consistent with the antagonist being able to bind to the resting state. Similar responses were observed in n = 6 independent cells. b Side view of rocuronium-bound structure, with subunits colored as in Fig. 1 and rocuronium as orange spheres. c Side view of two α subunits showing permeation pathway of b as dots representing solvent-accessible surface colored by diameter. Constriction points are formed by T244, L255 and L258, which are shown as sticks. e Rocuronium at α/γ interface. d Side view of rocuronium pore-blocked structure, with subunits colored as in Fig. 1 and rocuronium as orange spheres. e Rocuronium at the α/δ interface. f Plot of distance along pore axis vs. pore diameter for structures in this study compared to the apo receptor structure (PDB: 7SMM), ROC 1 -rocuronium-bound resting-like state, ROC 2 rocuronium-bound pore-blocked state. g Rocuronium at pore site. Ligands and interacting residues are shown as sticks and corresponding densities of ligands are shown as semitransparent surfaces.

Fig. 4. Agonist, blocker and anesthetic receptor sites and conformational changes.

ECDs are shown as black hollow ellipses, α_γ subunit TMD is in green, the TMD of α_δ subunit is shown in red, embedded in lipid bilayer. M4 helices of both subunits are shown as thick black lines between the TMD and lipid bilayer.