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Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor.

ABSTRACT: We examined functional consequences of intrasubunit contacts in the nicotinic receptor alpha subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues alphaK145, alphaD200, and alphaY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations alphaD200N and alphaK145Q impair channel gating to the same extent as either single mutation, while alphaK145E counteracts the impaired gating due to alphaD200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, alphaK145 and alphaD200 form a salt bridge associated with the closed state of the channel. When ACh binds, alphaY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches alphaK145, which in turn loosens its contact with alphaD200. The positional changes of alphaK145 and alphaD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of beta-strand 7, which harbors alphaK145 and is part of the signature Cys-loop, and the second is of beta-strand 10, which harbors alphaD200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel.

SUBMITTER: Mukhtasimova N

PROVIDER: S-EPMC2266616 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor.

Mukhtasimova Nuriya N Free Chris C Sine Steven M SM

The Journal of general physiology 20050613 1

We examined functional consequences of intrasubunit contacts in the nicotinic receptor alpha subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues alphaK145, alphaD200, and alphaY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, prim ...[more]

PMID: 15955875

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Project description:α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1β1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1β1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1β1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1β1δε nAChR, and showed loss of activity on α3β2 and α3β4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure-activity relationship (SAR) of EI revealed that the "Arg1-Asn2-Hyp3" residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1-3 EI caused a total loss of activity at the α1β1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1β1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1β1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.

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.

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Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor.

Publications

Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor.

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