Desensitization of mouse nicotinic acetylcholine receptor channels. A two-gate mechanism.
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ABSTRACT:
SUBMITTER: Auerbach A
PROVIDER: S-EPMC2525745 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The nicotinic acetylcholine receptor (nAChR) is a member of the important Cys loop ligand-gated ion channel superfamily that modulates neuronal excitability. After they respond to their agonists, their actions are terminated either by removal of ligand or by fast and slow desensitization, processes that play an important role in modulating the duration of conducting states and hence of integrated neuronal behavior. We monitored structural changes occurring during fast and slow desensitization in the transmembrane domain of the Torpedo nAChR using time-resolved photolabeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). After channel opening, TID photolabels a residue on the delta-subunit's M2-M3 loop and a cluster of four residues on deltaM1 and deltaM2, defining an open state pocket [Arevalo, E., et al. (2005) J. Biol. Chem. 280, 13631-13640]. We now find that photolabeling of this pocket persists during the transition to the fast desensitized state, the extent of photoincorporation decreasing only with the transition to the slow desensitized state. In contrast, the extent of photoincorporation in the channel lumen at the conserved 9'-leucines on the second transmembrane helix (M2-9') decreased successively during the resting to open and open to fast desensitized state transitions, implying that the local conformation is different in each state, a conclusion consistent with the hypothesis that there are separate gates for channel opening and desensitization. Thus, although during fast desensitization there is a conformation change in the channel lumen at the level of M2-9', there is none in the regions of the delta-subunit's M2-M3 loop and the interior of its M1-M4 helix bundle until slow desensitization occurs.
| S-EPMC2818735 | biostudies-literature
Project description:1. The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis. 2. The dissociation equilibrium constants were (mM): ACh (0. 16)<oxotremorine M (0.6)<carbamylcholine (0.8)<nicotine (2.6). 3. The gating equilibrium constants (opening/closing) were: ACh (45)>carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0. 5)>muscarine (0.15). 4. Rat neuronal alpha4beta2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined. 5. Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site.
| S-EPMC1571784 | biostudies-literature
Project description:Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for ?7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the ?7 nAChR.
| S-EPMC3137104 | biostudies-literature
Project description:Binding of the neurotransmitter acetylcholine to its receptors on muscle fibers depolarizes the membrane and thereby triggers muscle contraction. We sought to understand at the level of three-dimensional structure how agonists and antagonists alter nicotinic acetylcholine receptor conformation. We used the muscle-type receptor from the Torpedo ray to first define the structure of the receptor in a resting, activatable state. We then determined the receptor structure bound to the agonist carbachol, which stabilizes an asymmetric, closed channel desensitized state. We find conformational changes in a peripheral membrane helix are tied to recovery from desensitization. To probe mechanisms of antagonism, we obtained receptor structures with the active component of curare, a poison arrow toxin and precursor to modern muscle relaxants. d-Tubocurarine stabilizes the receptor in a desensitized-like state in the presence and absence of agonist. These findings define the transitions between resting and desensitized states and reveal divergent means by which antagonists block channel activity of the muscle-type nicotinic receptor.
| S-EPMC9531584 | biostudies-literature
Project description:PurposeThe α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse.MethodsTo examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.ResultsIn the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while β3 and β4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, β4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), β2, β4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species.ConclusionsRather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.
| S-EPMC4169779 | biostudies-literature
Project description:The ?7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate ?7 remains enigmatic. In particular, a major population of ?7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating ?7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of ?7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of ?7. Thus, calcium associates with a novel structural motif on ?7 and is an obligate cofactor in regions of limited ACh concentration.
| S-EPMC7478872 | biostudies-literature
Project description:The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.
| S-EPMC8135066 | biostudies-literature
Project description:PNU-120596 (1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea), a Type-II positive allosteric modulator of ?(7) nicotinic acetylcholine receptors inhibits ?(7) desensitization and robustly prolongs openings of ?(7) channels. However, these effects may render ?(7) channels more accessible to positively charged molecules and thus, more susceptible to voltage-dependent open-channel-block-like inhibition. To test this hypothesis, choline chloride (i.e., choline), a selective endogenous ?(7) agonist, and bicuculline methochloride (i.e., bicuculline), a competitive ?(7) antagonist, were used as membrane voltage-sensitive probes in whole-cell voltage-clamp recordings from hippocampal CA1 interneurons in acute brain slices in the absence and presence of PNU-120596. PNU-120596 enhanced voltage-dependent inhibition of ?(7) responses by bicuculline and choline. In the presence of PNU-120596, ?(7) channels favored a burst-like kinetic modality in the presence, but not absence of bicuculline and bursts of ?(7) openings were voltage-dependent. These results suggest that PNU-120596 alters the pharmacology of ?(7) channels by making these channels more susceptible to voltage-dependent inhibitory interactions with positively charged drugs at concentrations that do not potently inhibit ?(7) channels without PNU-120596. This inhibition imitates ?(7) nicotinic receptor desensitization and compromises the potentiating anti-desensitization effects of PNU-120596 on ?(7) nicotinic receptors. This unexpected dual action of PNU-120596, and possibly other Type-II positive allosteric modulators of ?(7) nicotinic receptors, may lead to unanticipated ?(7) channel-drug interactions and misinterpretation of ?(7) single-channel data.
| S-EPMC3839061 | biostudies-literature
Project description: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 reported that heterologously expressed nAChR containing α4 and β2χ subunits displayed a faster whole-cell current decay in its agonist response compared to responses of all-wild-type α4β2-nAChR. This suggests an unexpected, functional role for the C2 domain of the β2 subunit in α4β2-nAChR acute desensitization. Here we report that there also is faster desensitization of α4β4χ-nAChR relative to α4β4-nAChR stably and heterologously expressed in the human SH-EP1 cell-line. In addition, cell-attached, single-channel recording shows that both acetylcholine-activated α4β2χ- and α4β4χ-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 α4(∗)-nAChR containing chimeric instead of wild-type β subunits. Our findings also remain consistent with novel and unexpected roles of β subunit-nested C2 domains in modulation of α4(∗)-nAChR function.
| S-EPMC4344923 | biostudies-literature
Project description:A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.
| S-EPMC3381156 | biostudies-literature