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Roles of nicotinic acetylcholine receptor ? subunit cytoplasmic loops in acute desensitization and single-channel features.

ABSTRACT: To evaluate physiological roles of the large, second cytoplasmic loops (C2) situated between the M3 and M4 transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits. We have constructed chimeric ?2 (?2?) and ?4 (?4?) subunits in which the "nested" C2 domains (but not the "proximal" sequences of ?14 residues immediately adjacent to the M3 or M4 domains) of these ? subunits were replaced by the corresponding sequence from the serotonin 5-HT3A receptor subunit. We previously reported that heterologously expressed nAChR containing ?4 and ?2? subunits displayed a faster whole-cell current decay in its agonist response compared to responses of all-wild-type ?4?2-nAChR. This suggests an unexpected, functional role for the C2 domain of the ?2 subunit in ?4?2-nAChR acute desensitization. Here we report that there also is faster desensitization of ?4?4?-nAChR relative to ?4?4-nAChR stably and heterologously expressed in the human SH-EP1 cell-line. In addition, cell-attached, single-channel recording shows that both acetylcholine-activated ?4?2?- and ?4?4?-nAChR have a significantly lower mean open probability, shorter mean open-time, and a longer mean closed-time than their fully wild-type counterparts while not having different conductance amplitudes. These findings reveal microscopic bases for the faster desensitization of ?4(?)-nAChR containing chimeric instead of wild-type ? subunits. Our findings also remain consistent with novel and unexpected roles of ? subunit-nested C2 domains in modulation of ?4(?)-nAChR function.

SUBMITTER: Liu Q

PROVIDER: S-EPMC4344923 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Roles of nicotinic acetylcholine receptor β subunit cytoplasmic loops in acute desensitization and single-channel features.

Liu Q Q Kuo Y-P YP Shen J J Lukas R J RJ Wu J J

Neuroscience 20141220

To evaluate physiological roles of the large, second cytoplasmic loops (C2) situated between the M3 and M4 transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits. We have constructed chimeric β2 (β2χ) and β4 (β4χ) subunits in which the "nested" C2 domains (but not the "proximal" sequences of ∼14 residues immediately adjacent to the M3 or M4 domains) of these β subunits were replaced by the corresponding sequence from the serotonin 5-HT3A receptor subunit. We previously report ...[more]

PMID: 25536046

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Project description:The nicotinic acetylcholine receptor (nAChR) is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD) and steered rotation molecular dynamics (SRMD) simulations have been conducted on the cryo-electron microscopy (cryo-EM) structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC) bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh) binding pockets induced an inward and upward motion of the outer beta-sheet composed of beta9 and beta10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC), transmembrane (TM), and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA). To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O <--> C) motion. The present MD simulations explore the structural dynamics of the receptor under its gating process and provide a new insight into the gating mechanism of nAChR at the atomic level.

Project description:Background and purposeNeonicotinoid insecticides interact with the orthosteric site formed at subunit interfaces of insect nicotinic ACh (nACh) receptors. However, their interactions with the orthosteric sites at α-non α and α-α subunit interfaces remain poorly understood. The aim of this study was to elucidate the mechanism of neonicotinoid actions using the Drosophila Dα1-chicken β2 hybrid nACh receptor.Experimental approachComputer models of the (Dα1)3 (β2)2 nACh receptor in complex with imidacloprid and thiacloprid were generated. Amino acids in the Dα1 subunit were mutated to corresponding amino acids in the human α4 subunit to examine their effects on the agonist actions of neonicotinoids on (Dα1)3 (β2)2 and (Dα1)2 (β2)3 nACh receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology.Key resultsThe (Dα1)3 (β2)2 nACh receptor models indicated that amino acids in loops D, E and G probably determine the effects of neonicotinoids. The amino acid mutations tested had minimal effects on the EC50 for ACh. However, the R57S mutation in loop G, although having minimal effect on imidacloprid's actions, reduced the affinity of thiacloprid for the (Dα1)3 (β2)2 nACh receptor, while scarcely affecting thiacloprid's action on the (Dα1)2 (β2)3 nACh receptor. Both the K140T and the combined R57S;K140T mutations reduced neonicotinoid efficacy but only for the (Dα1)3 (β2)2 nACh receptor. Combining the E78K mutation with the R57S;K140T mutations resulted in a selective reduction of thiacloprid's affinity for the (Dα1)3 (β2)2 nACh receptor.Conclusions and implicationsThese findings suggest that a triangle of residues from loops D, E and G contribute to the selective actions of neonicotinoids on insect-vertebrate hybrid nACh receptors.Linked articlesThis article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Dataset Information

Roles of nicotinic acetylcholine receptor ? subunit cytoplasmic loops in acute desensitization and single-channel features.

Publications

Roles of nicotinic acetylcholine receptor β subunit cytoplasmic loops in acute desensitization and single-channel features.

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