Torpedo Acetylcholine Receptor Research Articles

A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor

Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit β1-β2 and M2-M3 loops, with agonist binding re-positioning a key β1-β2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9’ hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels.

Impact on AChR open channel noise by pore-peripheral salt bridge depends on voltage and divalent cations

Mechanisms behind the fluctuations in the ionic current through single acetylcholine receptor (AChR) channels have remained elusive. In a recent study of muscle AChR we showed that mutation of a conserved intramembrane salt bridge in the β- and δ-subunits markedly increased fluctuations in the open channel current that extended from low to high frequency. Here, we show that extracellular divalent cations reduce the high-frequency fluctuations and increase the low-frequency fluctuations. The low-frequency fluctuations are shown to arise from steps between two current levels, with the ratio of the time at each level changing e-fold for a 70 mV increase in membrane potential, indicating modulation by a charged element within the membrane field. Increasing the charge on the ion selectivity filter biases the ratio of current levels equivalent to a 50 mV increase in membrane potential but does not alter the voltage dependence of the ratio. The magnitudes of the voltage dependence and voltage bias allow estimates of the distance between the ion selectivity filter and the voltage-sensing element. Studies with either calcium or magnesium show that the two divalent cations synergize to increase the low-frequency fluctuations, whereas they act independently to decrease the high-frequency fluctuations, indicating multiple divalent cation binding sites. Molecular dynamics simulations applied to the structure of the Torpedo AChR reveal that mutation of the salt bridge alters the equilibrium positions and dynamics of residues local to the site of the mutation and within the adjacent ion selectivity filter in a calcium-dependent manner. Thus, disruption of a conserved intramembrane salt bridge in the muscle AChR induces fluctuations in open channel current that are sensitive to divalent cation binding at multiple sites and modulated by a charged element within the membrane field.

A Natural Variant of the Signaling Molecule Vav1 Enhances Susceptibility to Myasthenia Gravis and Influences the T Cell Receptor Repertoire.

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell receptor (TCR) signals and plays an important role in T cell development and activation. Previous genetic studies identified a natural variant of Vav1 characterized by the substitution of an arginine (R) residue by a tryptophane (W) at position 63 (Vav1R63W). This variant impacts Vav1 adaptor functions and controls susceptibility to T cell-mediated neuroinflammation. To assess the implication of this Vav1 variant on the susceptibility to antibody-mediated diseases, we used the animal model of myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). To this end, we generated a knock-in (KI) mouse model bearing a R to W substitution in the Vav1 gene (Vav1R63W) and immunized it with either torpedo acetylcholine receptor (tAChR) or the α146-162 immunodominant peptide. We observed that the Vav1R63W conferred increased susceptibility to EAMG, revealed by a higher AChR loss together with an increased production of effector cytokines (IFN-γ, IL-17A, GM-CSF) by antigen-specific CD4+ T cells, as well as an increased frequency of antigen-specific CD4+ T cells. This correlated with the emergence of a dominant antigen-specific T cell clone in KI mice that was not present in wild-type mice, suggesting an impact on thymic selection and/or a different clonal selection threshold following antigen encounter. Our results highlight the key role of Vav1 in the pathophysiology of EAMG and this was associated with an impact on the TCR repertoire of AChR reactive T lymphocytes.

Engineered agrin attenuates the severity of experimental autoimmune myasthenia gravis.

Agrin is essential for the formation and maintenance of neuromuscular junctions (NMJs). NT-1654 is a C-terminal fragment of mouse neural agrin. In this study, we determined the effects of NT-1654 on the severity of experimental autoimmune myasthenia gravis (EAMG). EAMG was induced in female Lewis rats by immunization with the Torpedo acetylcholine receptor (tAChR) and complete Freund's adjuvant (CFA). NT-1654 was dissolved in phosphate-buffered saline (PBS) and injected daily subcutaneously into tAChR immunized rats during the first 10 days after immunization, and then every other day for the following 20 days. We showed that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. We demonstrated that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. Muscle Nerve 57: 814-820, 2018.

Guidelines for standard preclinical experiments in the mouse model of myasthenia gravis induced by acetylcholine receptor immunization

Myasthenia gravis (MG) is an autoimmune disorder characterized by generalized muscle weakness due to neuromuscular junction (NMJ) dysfunction brought by acetylcholine receptor (AChR) antibodies in most cases. Although steroids and other immunosuppressants are effectively used for treatment of MG, these medications often cause severe side effects and a complete remission cannot be obtained in many cases. For pre-clinical evaluation of more effective and less toxic treatment methods for MG, the experimental autoimmune myasthenia gravis (EAMG) induced by Torpedo AChR immunization has become one of the standard animal models. Although numerous compounds have been recently proposed for MG mostly by using the active immunization EAMG model, only a few have been proven to be effective in MG patients. The variability in the experimental design, immunization methods and outcome measurements of pre-clinical EAMG studies make it difficult to interpret the published reports and assess the potential for application to MG patients. In an effort to standardize the active immunization EAMG model, we propose standard procedures for animal care conditions, sampling and randomization of mice, experimental design and outcome measures. Utilization of these standard procedures might improve the power of pre-clinical EAMG experiments and increase the chances for identifying promising novel treatment methods that can be effectively translated into clinical trials for MG.

Delivery of an miR155 inhibitor by anti-CD20 single-chain antibody into B cells reduces the acetylcholine receptor-specific autoantibodies and ameliorates experimental autoimmune myasthenia gravis

MicroRNA-155 (miR155) is required for antibody production after vaccination with attenuated Salmonella. miR155-deficient B cells generated reduced germinal centre responses and failed to produce high-affinity immunoglobulin (Ig)G1 antibodies. In this study, we observed up-regulation of miR155 in the peripheral blood mononuclear cells (PBMCs) of patients with myasthenia gravis (MG), and miR155 was also up-regulated in torpedo acetylcholine receptor (T-AChR)-stimulated B cells. We used an inhibitor of miR155 conjugated to anti-CD20 single-chain antibody to treat both the cultured B cells and the experimental autoimmune MG (EAMG) mice. Our results demonstrated that silencing of miR155 by its inhibitor impaired the B cell-activating factor (BAFF)-R-related signalling pathway and reduced the translocation of nuclear factor (NF)-κB into the nucleus. Additionally, AChR-specific autoantibodies were reduced, which may be related to the altered amounts of marginal zone B cells and memory B cells in the spleens of EAMG mice. Our study suggests that miR155 may be a promising target for the clinical therapy of MG.

Interaction of nicotinic receptors with bupropion: Structural, functional, and pre-clinical perspectives

Besides the antidepressant activity of bupropion (BP), preclinical studies in rodents provide evidence that this compound and its hydroxyl metabolites can attenuate nicotine withdrawal, reversing both the physical and negative affective aspects of nicotine abstinence. Co-interactions of BP with nicotine or other psychostimulants influence decrease anxiety- and cognitive- related processes. BP also attenuates the reinstatement of nicotine-induced conditioned place preference in rats caused by a priming dose of nicotine, morphine, cannabinoids, or ethanol. Therefore, BP can offer an interesting approach to the prevention of relapse in humans. There is emerging evidence that BP inhibits, in the low to intermediate micromolar range, various nicotinic acetylcholine receptors (AChRs) expressed in different neuronal pathways. The BP selectivity for different AChRs follows the sequence: α3- > α4- ~ α1- > α7-containing AChRs. This receptor blockade may contribute to its dual therapeutic activity as either an antidepressant or anti-nicotinic drug. Regarding the structural aspects, two distinct binding sites for [125I]SADU-3-72, a photosensitive derivative of BP, were identified in the Torpedo AChR. A binding site is located within the ion channel, which coincides with the molecular docking results, whereas a second site is found near the extracellular end of α1-M1 when the receptor is in the desensitized state. Interestingly, BP greatly reduces the potentiating action elicited by Zn2+ on different non-α7 AChRs, and vice versa this cation increases the inhibitory strength of BP. This contrasting behavior supports the concept that the binding sites for BP and Zn2+are located at different domains. The understanding of the BP activity, alone or in combination with other drugs (e.g., nicotine), at the molecular and behavioral levels, may improve the knowledge of the underlying mechanisms of action. This knowledge is essential for the development of novel BP (or other antidepressant) derivatives with improved clinical profiles for the treatment of depression and psychostimulant-related addictions. Normal 0 7.8 磅 0 2 false false false MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:普通表格; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Times New Roman; mso-fareast-font-family:Times New Roman; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;}

Design, synthesis, and characterization of a 39 amino acid peptide mimic of the main immunogenic region of the Torpedo acetylcholine receptor

We have designed a 39 amino acid peptide mimic of the conformation-dependent main immunogenic region (MIR) of the Torpedo acetylcholine receptor (TAChR) that joins three discontinuous segments of the Torpedo α-subunit, α(1–12), α(65–79), and α(110 – 115) with two GS linkers:123456789101112656667686970717273747576777879110 – 115SEHETRLVANLLGGGSLRWNPADYGGIKKIRGSLDYTGKThis 39MIR-mimic was expressed in E. coli as a fusion protein with an intein–chitin-binding domain (IChBD) to permit affinity collection on chitin beads. Six MIR-directed monoclonal antibodies (mAbs) bind to this complex and five agonist/antagonist site directed mAbs do not. The complex of MIR-directed mAb-132A with 39MIR has a Kd of (2.11±0.11)×10−10M, which is smaller than (7.13±1.20)×10−10M for the complex of mAb-132A with α(1–161) and about the same as 3.4×10−10M for that of mAb-132A with TAChR. Additionally, the 39MIR-IChBD adsorbs all MIR-directed antibodies (Abs) from an experimental autoimmune myasthenia gravis (EAMG) rat serum. Hence, the 39MIR-mimic has the potential to inactivate or remove pathogenic Torpedo MIR-directed Abs from EAMG sera and to direct a magic bullet to the memory B-cells that produce those pathogenic Abs. The hope is to use this as a guide to produce a mimic of the human MIR on the way to an antigen specific therapeutic agent to treat MG.

Structural characterization of the main immunogenic region of the Torpedo acetylcholine receptor

To develop antigen-specific immunotherapies for autoimmune diseases, knowledge of the molecular structure of targeted immunological hotspots will guide the production of reagents to inhibit and halt production of antigen specific attack agents. To this end we have identified three noncontiguous segments of the Torpedo nicotinic acetylcholine receptor (AChR) α-subunit that contribute to the conformationally sensitive immunological hotspot on the AChR termed the main immunogenic region (MIR): α(1–12), α(65–79), and α(110–115). This region is the target of greater than 50% of the anti-AChR Abs in serum from patients with myasthenia gravis (MG) and animals with experimental autoimmune myasthenia gravis (EAMG). Many monoclonal antibodies (mAbs) raised in one species against an electric organ AChR cross react with the neuromuscular AChR MIR in several species. Probing the Torpedo AChR α-subunit with mAb 132A, a disease inducing anti-MIR mAb raised against the Torpedo AChR, we have determined that two of the three MIR segments, α(1–12) and α(65–79), form a complex providing the signature components recognized by mAb 132A. These two segments straddle a third, α(110–115), that seems not to contribute specific side chains for 132A recognition, but is necessary for optimum antibody binding. This third segment appears to form a foundation upon which the three-dimensional 132A epitope is anchored.

Assessment of Homology Templates and an Anesthetic Binding Site within the γ-Aminobutyric Acid Receptor

Anesthetics mediate portions of their activity via modulation of the γ-aminobutyric acid receptor (GABAaR). Although its molecular structure remains unknown, significant progress has been made toward understanding its interactions with anesthetics via molecular modeling. The structure of the torpedo acetylcholine receptor (nAChRα), the structures of the α4 and β2 subunits of the human nAChR, the structures of the eukaryotic glutamate-gated chloride channel (GluCl), and the prokaryotic pH-sensing channels, from Gloeobacter violaceus and Erwinia chrysanthemi, were aligned with the SAlign and 3DMA algorithms. A multiple sequence alignment from these structures and those of the GABAaR was performed with ClustalW. The Modeler and Rosetta algorithms independently created three-dimensional constructs of the GABAaR from the GluCl template. The CDocker algorithm docked a congeneric series of propofol derivatives into the binding pocket and scored calculated binding affinities for correlation with known GABAaR potentiation EC50s. Multiple structure alignments of templates revealed a clear consensus of residue locations relevant to anesthetic effects except for torpedo nAChR. Within the GABAaR models generated from GluCl, the residues notable for modulating anesthetic action within transmembrane segments 1, 2, and 3 converged on the intersubunit interface between α and β subunits. Docking scores of a propofol derivative series into this binding site showed strong linear correlation with GABAaR potentiation EC50. Consensus structural alignment based on homologous templates revealed an intersubunit anesthetic binding cavity within the transmembrane domain of the GABAaR, which showed a correlation of ligand docking scores with experimentally measured GABAaR potentiation.

(−)-Reboxetine inhibits muscle nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites

The interaction of (−)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (−)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca2+ influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC50=3.86±0.49 and 1.92±0.48μM, respectively, (b) binds to the [3H]TCP site with ∼13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [3H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6′ and 13′ in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9′ is the minimum structural component for (−)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (−)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants.

Nicotinic Receptor Transduction Zone: Invariant Arginine Couples to Multiple Electron-Rich Residues

Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from β-strand 10 of the binding domain, the invariant αArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of αArg209 to channel gating depends strongly on αGlu45, also within this region. Here we determine whether the contribution of αArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of αArg209 on αGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of αArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, αArg209 and αGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation αE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains.

Structural Analysis and Ion Translocation Mechanisms of the Muscle-Type Acetylcholine Receptor Channel

The aim of this work is to analyze the conformational changes in the acetylcholine receptor caused by channel opening and to investigate the electrostatic profile during ion translocation through the channel. A computational model of the human muscle-type acetylcholine receptor (AChR) was built and used to analyze channel structure and its interactions with different ions. Using the Torpedo AChR crystal structure as a homologous template, the 3D structure of the human muscle-type AChR was reconstructed. This first model is optimized and an open structure of the channel is generated using Normal Mode Analysis in order to assess morphologic and energetic differences between open and closed structures. In addition, the issue of ion translocation is investigated in further detail. Results elucidate different aspects of the channel: channel gate structure, channel interactions with translocating ions, differences between muscle-type AChR and previous neuronal-type AChR models. The model constructed here is ideal for further computational studies on muscle-type AChR and its pathologic mutations.

Structural and functional interaction of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone, a photoreactive bupropion derivative, with nicotinic acetylcholine receptors

The pharmacological properties of (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca2+ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [3H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13′) and serine (position 6′) rings. However, an additional domain, between the outer (position 20′) and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel.

Peptide mimics of the torpedo and human AChR main immunogenic regions

Peptide mimics of the torpedo and human AChR main immunogenic regions

Molecular Interactions between Ligands and Nicotinic Acetylcholine Receptors Revealed by Studies with Acetylcholine Binding Proteins

Nicotinic acetylcholine receptors (AChRs) are the best characterized ion channels representing the Cys-loop ligand-gated ion channel superfamily. Studies using Torpedo AChRs in the closed and open states andacetylcholine binding proteins (AChBPs) from different origins have elucidated the most important structural and functional features of the agonist/competitive antagonistbinding sites. The first step in recognizing the neurotransmitter ACh and other agonists is fundamental in the process of agonist-induced activation, including the opening of the intrinsic cation channel. The AChBP studies demonstrated that Loop C is an important structural feature that is modified by ligand binding. These studies defined important pharmacologic features of AChR ligands, including the differences between full and partial agonists, agonists and competitive antagonists, peptidic and non-peptidic ligands, and between high affinity and high selectivity. The studies showing the structural mechanisms by which specific ligands can activate, inhibit, and potentiate different AChR subtypes could be of therapeutic importance.

Agonist Interactions and Selectivity in Inhibitory Cys-Loop Ligand-Gated Ion Channels

Cys-loop ligand-gated ion channels constitute one of two major superfamilies of receptors mediating rapid chemical synaptic transmission in the central nervous system. Mammalian members of the superfamily include cation selective channels that are receptors for excitatory neurotransmitters, acetylcholine and serotonin, and anion selective channels that are receptors for inhibitory neurotransmitters, GABA and glycine. Structural information from snail acetylcholine binding proteins (AChBP), torpedo acetylcholine receptors provided a clear picture of the acetylcholine-binding site, including the conserved aromatic box which forms cation-pi bonds with bound agonists. From AChBP-based homology models, we proposed (Cromer 2002) that the 3-dimensional position of one of the aromatic box residues is replaced with an acidic residue in inhibitory receptors for GABA (GABAA/CR) and glycine (GlyR). Further that this residue forms a salt-bridge interaction with the cation group of bound agonists and is important for selectivity for primary amine agonists, such as GABA and glycine, over bulkier quaternary amines, such as acetylcholine.We now present evidence in support of this hypothesis, particularly using the homopentameric Rho1 GABA receptor (GABAC) as a model for the broader family of inhibitory Cys-loop LGICs. We also present evidence for a series of other interactions in the agonist-binding site of inhibitory Cys-loop LGICs that are determinants of selectivity between agonists of different size, such as glycine versus GABA. These results are consistent with our initial hypothesis and provide a more detailed understanding of agonist-receptor interactions in inhibitory Cys-loop ligand-gated ion channels.Cromer, B. A., Morton, C. J. & Parker, M. W. (2002). Anxiety over GABA(A) receptor structure relieved by AChBP. Trends Biochem Sci 27, 280-7.

Structure–activity relationship of ibogaine analogs interacting with nicotinic acetylcholine receptors in different conformational states

The interaction of ibogaine analogs with nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that ibogaine analogs: (a) inhibit (±)-epibatidine-induced Ca 2+ influx in human embryonic muscle AChRs with the following potency sequence (IC 50 in μM): (±)-18-methylaminocoronaridine (5.9 ± 0.3) ∼ (±)-18-methoxycoronaridine (18-MC) (6.8 ± 0.8) > (−)-ibogaine (17 ± 3) ∼ (+)-catharanthine (20 ± 1) > (±)-albifloranine (46 ± 13), (b) bind to the [ 3H]TCP binding site with higher affinity when the Torpedo AChR is in the desensitized state compared to that in the resting state. Similar results were obtained using [ 3H]18-MC. These and docking results suggest a steric interaction between TCP and ibogaine analogs for the same site, (c) enhance [ 3H]cytisine binding to resting but not to desensitized AChRs, with desensitizing potencies (apparent EC 50) that correlate very well with the p K i values in the desensitized state, and (d) there are good bilinear correlations between the ligand molecular volumes and their affinities in the desensitized and resting states, with an optimal volume of ∼345 Å 3 for the ibogaine site. These results indicate that the size of the binding sites for ibogaine analogs, located between the serine and nonpolar rings and shared with TCP, is an important structural feature for binding and for inducing desensitization.

Novel Positive Allosteric Modulators of the Human α7 Nicotinic Acetylcholine Receptor

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ∼12 Å from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.

Proteasome Inhibition with Bortezomib Depletes Plasma Cells and Autoantibodies in Experimental Autoimmune Myasthenia Gravis

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.