Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy.
Ontology highlight
ABSTRACT:
SUBMITTER: Opella SJ
PROVIDER: S-EPMC3282055 | biostudies-literature | 1999 Apr
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel in the postsynaptic membrane. It is composed of five homologous subunits, each of which contributes one transmembrane helix--the M2 helix--to create the channel pore. The M2 helix from the delta subunit is capable of forming a channel by itself. Although a model of the receptor was recently proposed based on a low-resolution, cryo-electron microscopy density map, we found that the model does not explain much of the other available experimental data. Here we propose a new model of the M2 channel derived solely from helix packing and symmetry constraints. This model agrees well with experimental results from solid-state NMR, chemical reactivity, and mutagenesis experiments. The model depicts the channel pore, the channel gate, and the residues responsible for cation specificity.
| S-EPMC1304489 | biostudies-literature
Project description:Over the past four decades, the patch clamp technique and nicotinic ACh (nACh) receptors have established an enduring partnership. Like all good partnerships, each partner has proven significant in its own right, while their union has spurred innumerable advances in life science research. A member and prototype of the superfamily of pentameric ligand-gated ion channels, the nACh receptor is a chemo-electric transducer, binding ACh released from nerves and rapidly opening its channel to cation flow to elicit cellular excitation. A subject of a Nobel Prize in Physiology or Medicine, the patch clamp technique provides unprecedented resolution of currents through single ion channels in their native cellular environments. Here, focusing on muscle and α7 nACh receptors, we describe the extraordinary contribution of the patch clamp technique towards understanding how they activate in response to neurotransmitter, how subtle structural and mechanistic differences among nACh receptor subtypes translate into significant physiological differences, and how nACh receptors are being exploited as therapeutic drug targets.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/.
| S-EPMC5979820 | biostudies-literature
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.
| S-EPMC2211534 | biostudies-literature
Project description:The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH while W41 ensures proton conduction only from the N terminus to the C terminus and prevents reverse current under low internal pH. Here, we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low internal pH. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type (WT) channel but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted toward the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C terminus. At low pH, the mutant channel contains more cationic tetrads than the WT channel, consistent with faster reverse protonation from the C terminus. 15N NMR spectra allow the extraction of four H37 pKas and show that the pKas are more clustered in the mutant channel compared to WT M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in WT M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in WT M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants.
| S-EPMC5761075 | biostudies-literature
Project description:Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the ?4?2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(?4?2)2?2- and LS-(?4?2)2?4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity ?4(+)/(-)?2 subunit binding interfaces. However LS-(?4?2)2?4-nAChR also respond to higher concentrations of ACh, acting at a third ?4(+)/(-)?4 subunit interface. To probe isoform functional differences further, HS- and LS-?4?2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(?4?2)2?2-nAChR exhibit a single conductance state, whereas LS-(?4?2)2?4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(?4?2)2?4-nAChR bursting and reduced closed times. Introduction of an ?4(+)/(-)?4-interface loss-of-function ?4W182A mutation abolished these changes, confirming this site's role in mediating LS-(?4?2)2?4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(?4?2)2?4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by ?4(+)/(-)?4 site ACh binding. These findings are consistent with ?4(+)/(-)?4 subunit interface occupation resulting in allosteric potentiation of agonist actions at ?4(+)/(-)?2 subunit interfaces, rather than independent induction of high conductance channel openings.
| S-EPMC6405073 | biostudies-literature
Project description:The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.
| S-EPMC8831596 | biostudies-literature
Project description:Construction of a GABAA receptor homology model based on the acetylcholine (ACh) receptor structure is complicated by the low sequence similarity between GABAA and ACh M3 transmembrane segments that creates significant uncertainty in their alignment. We determined the orientation of the GABAA M2 and M3 transmembrane segments using disulfide cross-linking. The M2 residues alpha1M266 (11') and alpha1T267 (12') were mutated to cysteine in either wild type or single M3 cysteine mutant (alpha1V297C, alpha1A300C to alpha1A305C) backgrounds. We assayed spontaneous and induced disulfide bond formation. Reduction with DTT significantly potentiated GABA-induced currents in alpha1T267C-L301C and alpha1T267C-F304C. Copper phenanthroline-induced oxidation inhibited GABA-induced currents in these mutants and in alpha1T267C-A305C. Intrasubunit disulfide bonds formed between these Cys pairs, implying that the alpha-carbon separation was at most 5.6 A. The reactive alpha1M3 residues (L301, F304, A305) lie on the same face of an alpha-helix. The unresponsive ones (A300, I302, E303) lie on the opposite face. In the resting state, the reactive side of alpha1M3 faces M2-alpha1T267. In conjunction with the ACh structure, our data indicate that alignment of GABAA and ACh M3 requires a single gap in the GABAA M2-M3 loop. In the presence of GABA, oxidation of alpha1T267C-L301C and alpha1T267C-F304C had no effect, but oxidation of alpha1T267C-A305C caused a significant increase in spontaneous channel opening. We infer that, as the channel opens, the distance and/or orientation between M2-alpha1T267 and M3-alpha1A305 changes such that the disulfide bond stabilizes the open state. This begins to define the conformational motion that M2 undergoes during channel opening.
| S-EPMC6674078 | biostudies-literature
Project description:We studied single-channel currents from neuromuscular acetylcholine receptor-channels with mutations in the pore-lining, M2 helix of the epsilon-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E(2)), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E(2) on a log-log scale (Phi), which illuminates the energy character of the residue (C- versus O-like) within the C<-->O isomerization process; and 3), the open-channel current amplitude (i(0)), which reports whether a mutation alters the energetics of ion permeation. The largest E(2) changes were observed in the cytoplasmic half of epsilonM2 (5', 9', 12', 13', and 16'), with smaller changes apparent for residues > or =17'. Phi was approximately 0.54 for most epsilonM2 residues, but was approximately 0.32 at the positions that had largest E(2) changes. An arginine substitution reduced i(0) significantly at six positions, with the magnitude of the reduction increasing, 16'-->2'. The measurements suggest that the 9', 12', and 13' residues experience large and late free-energy changes in the channel-opening process. We speculate that in the gating isomerization the pore-facing residues >6' and <16' experience multiple energy perturbations associated with changes in protein structure and, perhaps, hydration.
| S-EPMC2712209 | biostudies-literature
Project description:Muscarinic acetylcholine receptors are G protein-coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes (M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the Gq/ 11 family, whereas M2R and M4R couple to the Gi/ o family. Here, we present and compare the cryo-electron microscopy structures of M1R in complex with G11 and M2R in complex with GoA The M1R-G11 complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed analysis of these structures provides a framework for understanding the molecular determinants of G-protein coupling selectivity.
| S-EPMC7034192 | biostudies-literature
Project description:Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4β2, but not α6 subunits. Polyamines are classically studied as regulators of ion channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology.
| S-EPMC7271128 | biostudies-literature