Top PDF Chemical-scale studies of the nicotinic and muscarinic acetylcholine receptors

Chemical-scale studies of the nicotinic and muscarinic acetylcholine receptors

Chemical-scale studies of the nicotinic and muscarinic acetylcholine receptors

In Chapter 2, a highly conserved aspartate residue (D89) that is near the agonist binding site of the nAChR was probed for its role in agonist binding. We found that the side chain of D89 establishes a redundant network of hydrogen bonds and preorganizes the agonist binding site by positioning a critical agonist-binding residue, tryptophan 149 (W149). Previous studies of a D89N mutant led to the proposal that a negative charge at D89 was essential for receptor function. However, our studies show that neutral side chains at position 89 function well, only if an unfavorable electrostatic clash is avoided.
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Chemical-Scale Studies of the Nicotinic and Muscarinic Acetylcholine Receptors

Chemical-Scale Studies of the Nicotinic and Muscarinic Acetylcholine Receptors

the fluorinated Trp series simultaneously at both residues should present the expected linear relationship between receptor response and cation-π binding energy. 3.3.4 Other Possible Cation- π Interaction Sites and Future M 2 AChR Experiments The recent crystal structure of the β 2 AR with the inverse agonist carazolol bound provides some possible sites of interest for future studies on the M 2 AChR 31 . There are nine residues found within 5 Å of the ligand that are aromatic residues in M 2 AChR (Figure 3.27); we have studied three of these residues in this project. Of the six remaining aromatic residues, only one, W7.35, is a Trp. Would the quaternary amine of ACh bind to a Tyr or Phe instead of a Trp, even though Trp is the stronger cation-π binder? In previous experiments on the α7 nAChR, a cation-π interaction was discovered at Tyr 93 even though the classic Trp 149 was also present 81 . So, there is precedent for such a Tyr cation-π site. In fact, a recent model of the M 1 AChR suggested that residues Y7.39, Y6.51, and Y7.43 are in the same proximity to the quaternary amine of ACh as the aromatic box residues in the nAChR 36 . An Asn at position 7.39 in the β 2 AR appears to hydrogen bond with the secondary amine of carazolol in the crystal structure 31 . Therefore, there is evidence that a Tyr in the M 2 AChR may serve as the anchor point for the quaternary amine of ACh.
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Nicotinic acetylcholine receptors modulate osteoclastogenesis

Nicotinic acetylcholine receptors modulate osteoclastogenesis

Conclusions Our results suggest that cholinergic agonists inhibit RANKL-induced osteclastogenesis by interfering with intracellular calcium levels and consequently with the NFATc1 signaling pathway. Of note, this complete block- ade of osteoclastogenesis in vitro was neither due to toxic effects nor associated with apoptosis, and was shown to be reversible. However, while high levels of nAChR activity appear to inhibit RANKL-induced osteo- clastogenesis, actions mediated by certain nAChRs, in particular the α 7 homomeric receptor, may favor it. Fur- ther studies are needed to determine the gender-specific effect of α 7 and other nAChRs and mAChRs on bone homeostasis.
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Nicotinic acetylcholine receptors modulate osteoclastogenesis

Nicotinic acetylcholine receptors modulate osteoclastogenesis

Herein we demonstrate that nAChRs are crucially in- volved in RANKL-induced osteoclastogenesis by inhibit- ing Ca 2+ oscillations, which in turn blocks RANKL- induced activation of c-fos and NFATc1. In contrast to our findings, it has been recently suggested that nAChR agonists have no effect on osteoclastogenesis in vitro [12]; however, the ligands were used at lower concentra- tions than we utilized in our study. Tanaka et al. admin- istered nicotine at doses comparable to those used here, and observed a reduction in the number of large OCs and the resorptive index, but an increase in the number of small OCs suggesting a reduction in fusion [17]. We were also able to observe an increase in the number of osteoclasts upon administration of low-dose nicotine and PNU282987. Thus nicotinic agonists at low concen- tration may favor the generation of osteoclasts, whereas high concentrations inhibit osteoclastogenesis. Interest- ingly it has been suggested that different nAChRs may also convey opposing effects as evidenced by recent studies, which described increased in vitro osteoclasto- genesis as well as a significant, though modest, decrease in the systemic bone mass of α2–/– mice [12] and in- creased bending stiffness and cortical thickness in female α7–/– mice, while male counterparts showed no
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Lynx1 Modulation of Nicotinic Acetylcholine Receptors

Lynx1 Modulation of Nicotinic Acetylcholine Receptors

these α6L9’S mice and α-CTX MII, a toxin that specifically blocks α6* nicotinic receptors, we thought that we had good tools to study the effect of lynx1 on α6. Once we crossed the lynx1KO mice to the α6L9’S mice, we began behavioral studies. We examined their ability to habituate to novel environments and their home cage activity. We immediately observed that there were very few lynx1KO α6L9’S mice that were hyperactive, and it appeared that these mice did habituate to a novel environment. However, we did not properly account for the bimodal distribution of the α6L9’S mice in analyzing the results of the experiment. In fact, only about 50% of the α6L9’S mice are hyperactive (Drenan et al., 2008).
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Lophotoxin insensitive nematode nicotinic acetylcholine receptors

Lophotoxin insensitive nematode nicotinic acetylcholine receptors

JEB0417 Nematode nicotinic acetylcholine receptors (nAChRs) are molecular targets of several anthelmintic drugs. Studies to date on Caenorhabditis elegans and Ascaris suum have demonstrated atypical pharmacology with respect to nAChR antagonists, including the finding that κ-bungarotoxin is a more effective antagonist than α- bungarotoxin on Ascaris muscle nAChRs. Lophotoxin and its naturally occurring analogue bipinnatin B block all vertebrate and invertebrate nAChRs so far examined. In the present study, the effects on nematode nAChRs of bipinnatin B have been examined. The Ascaris suum muscle cell nAChR was found to be insensitive to 30 µmol l −1 bipinnatin B, a concentration that is highly effective on other nAChRs. To our knowledge, this is the first demonstration of a nAChR that is insensitive to one of the lophotoxins. Xenopus laevis oocytes injected with C.
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The assembly and folding of neuronal nicotinic acetylcholine receptors

The assembly and folding of neuronal nicotinic acetylcholine receptors

assembly, to some extent, with both rat a and P subunits. When the Drosophila ARD subunit is co-expressed with rat nAChR subunits there appears to be a rapid degradation of the rat nAChR subunits, which may indicate the production of a misfolded subunit complex. When the Drosophila ARD subunits was co-expressed with either the rat a 4 or p2 subunit, little or no a 4 or p2 subunit was revealed in the immunoprecipitates. It is possible therefore that the ARD can co- assemble with both rat a4 and p2 subunits, but the subunits are degraded owing to the production of incompletely folded protein complexes. This interpretation is supported by immunoprécipitation studies showing that when either rat a 4 or p2 are expressed alone, they can be detected at levels similar when they are co-expressed with subunits other than ARD. There is evidence from the expression of muscle nAChR subunits that misfolded subunit complexes do not exit from the ER but are rapidly degraded (reviewed by Green and Millar 1995). The band corresponding to the ARD protein in the co-immunoprecipiation studies (figures 5.6 - 5.9) runs at a size approximately 48 kD which is considerably smaller than the predicted size of of the mature protein which is approximately 58 kD. The size difference observed when ARD or the epitope-tagged ARD subunit is co-expressed with other subunits may be explained by partial degradation of the ARD protein.
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Chemical scale investigations of drug receptor interactions at the nicotinic acetylcholine receptor

Chemical scale investigations of drug receptor interactions at the nicotinic acetylcholine receptor

19 2.1 INTRODUCTION Biological signaling pathways employ a vast array of integral membrane proteins that process and interpret the chemical, electrical, and mechanical signals that are delivered to cells. These proteins are the targets of most drugs of therapy and abuse, but structural insights are sparse because both x-ray crystallography and NMR spectroscopy are of limited applicability. Even when structural information is available, establishing the functional importance of particular structural features can be challenging. In contrast, chemistry-based methods hold great promise for producing high-precision structural and functional insights. Varying the drug or signaling molecule has been the approach of the pharmaceutical industry, producing a multitude of structure-activity relationships of considerable value. In recent years we have taken the reverse approach, in which we systematically vary the receptor and use functional assays to monitor changes in drug- receptor interactions. 1,2 We show here that this physical chemistry approach to studying receptors can produce high-precision insights into drug-receptor interactions. In particular, we show that two agonists that interact with the same binding pocket of a receptor can make use of very different noncovalent interactions to achieve the same result.
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Binding Studies of Neuronal Nicotinic Acetylcholine Receptors Expressing Unnatural Amino Acids

Binding Studies of Neuronal Nicotinic Acetylcholine Receptors Expressing Unnatural Amino Acids

4.1 Introduction We have found that the smoking cessation drug, varenicline, acts on its target receptor, α4β2, via two binding interactions. First, a cation - π interaction to a conserved tryptophan residue (TrpB, Trp154 in rat α4) and second, a hydrogen bond to the backbone CO of TrpB. 1 As described in Chapter 2, we established that this binding pattern holds for both stoichio metries of the α4β2 receptor, A3B2 and A2B3. We also find that varenicline appears to violate the nicotinic pharmacophore by failing to make a functionally significant additional hydrogen bond to the backbone NH of a conserved leucine residue (L119 in rat β2). We have proposed that this is due to varenicline having a weaker hydrogen bond acceptor moiety than the other nicotinic agonists studied (acetylcholine, nicotine and cytisine). Studies described in Chapter 3 support this claim. The work presented in this chapter is aimed at further studying activation by varenicline of wild type and unnatural amino acid-expressing forms of A2B3 and A3B2 receptors. Specifically, we used single- channel recording with varenicline to determine if the gating properties of the receptor change upon fluorination of TrpB. Ideally, the best fluorinated TrpB mutant to study would be F 4 W. However, we find that F 4 W does not incorporate efficiently enough to provide the level of expression necessary for single-channel recording.
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Structure-function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids

Structure-function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids

2.1 Abstract In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high-precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a re-examination of previously reported data on γ-aminobutyric acid, type A, and glycine receptors, we concluded that the
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Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids

Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids

We also establish that a point mutation just four residues away from TrpB appears to influence the shape of the agonist binding site, such that it can differentiate the agonist binding mode of the α4β2 and muscle-type receptors. Chapter 3 extends studies of the point mutation near TrpB, termed the “loop B glycine.” We examine the muscle-type, α4β2, and α7 subtypes and show that the identity of this residue strongly correlates with agonist potency. Low-potency receptor subtypes have a glycine at the loop B site, while high-potency receptors have a lysine at this site. We establish that mutation of this residue can to convert a low-potency receptor to a high-potency receptor and vice versa.
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Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors

Effects of TI-299423 on Neuronal Nicotinic Acetylcholine Receptors

Other studies have shown that any history of smoking at all decreases the probability of developing PD in the future [10, 49]. The mechanism by which nicotine is proposed to be neuroprotective against PD seems to be associated directly with nicotine’s activation of nAChRs on dopaminergic neurons, and the long-term potentiation of those connections that can follow. In fact, previous studies have suggested that α6β2*-nAChRs, which are primarily responsible for nicotine induced dopamine release [47], may be necessary for neuroprotection, as their presence is only observed when nicotine neuroprotection has occurred [47]. This suggests that ligands targeting α6β2*-nAChRs and, to a lesser extent, α4β2*-nAChRs in the SNc could be used as potential treatments for the prevention of PD. Nicotine also reduces L-DOPA-induced dyskinesias [47]. Though the exact nAChR subtypes responsible for that are unknown, the prevalence of α6β2* and α4β2*-nAChRs in the brain implicates them as possible targets for this as well.
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Molecular mechanisms of allosteric modulation of nicotinic acetylcholine receptors

Molecular mechanisms of allosteric modulation of nicotinic acetylcholine receptors

However, several important questions about gating remain unanswered. Functional studies of slow onset desensitization have suggested the presence of a separate desensitization gate that extends from the -3’ to 9’ positions [Karlin 2002; Paas et al. 2005]. As all of the previously described computational simulations were on closed state receptor models, the conformational changes associated from the open state to the desensitized state are unknown. Since desensitization of nicotinic receptors increases proportionally to the number of binding sites [Rayes et al. 2009], homology models based on the heteromeric Torpedo nAChR may not accurately capture conformational changes associated with desensitization of the homomeric α 7 receptor. Our validated open state model will be an excellent tool to study open to desensitized conformational and electrostatic changes.
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Nicotinic acetylcholine receptors: specific antibodies and functions in humoral immunity

Nicotinic acetylcholine receptors: specific antibodies and functions in humoral immunity

play an important role in humoral immunity. Be- ing expressed in B lymphocytes, they regulate both the formation of their antigen-specific repertoire in the course of development and activation in mature state. Different nAChR subtypes are present along with the B lymphocyte maturation: α4β2 nAChRs are prevalent in immature B lymphocytes within the bone marrow, while α7 nAChRs are mostly found in mature B lymphocytes in the spleen. Possibly, this difference corresponds to different kinds of nAChR agonists present in the primary vs secon dary lymphoid organs. The bone marrow is innervated with cholinergic nerve fibers; therefore, differentiation of the blood cells can be regulated with neuronal acetylcholine through α4β2 nAChRs sensitive to nanomolar doses of this agonist. In contrast, the spleen has no cholinergic innerva- tion; therefore, the nAChRs expressed in mature B lymphocytes can be stimulated (or desensitized) either by choline, which is an α7-specific agonist, or by acetylcholine produced by activated T and B lymphocytes and affecting them locally within the immune synapse. α4β2 nAChRs are localized in close proximity to the antigen-specific receptor (BCR) and positively regulate its involvement in B lymphocyte differentiation. α7 nAChRs are located close to CD40 and negatively regulate its signal- ing in the course of B lymphocyte activation. α9 nAChRs comprise a minor population capable of fulfilling functions similar to those of α7 nAChRs and partially substitute them in α7-/- animals. on the whole, the data obtained delineate the role of cholinergic mechanisms in humoral immunity un- derlying the observed immunosuppression in smok- ers constantly consuming nAChR agonist nicotine. one of the ways through which the nAChRs are involved in B lymphocyte differentiation is the support of cell survival. our data have re- vealed that, in addition to established pro-survival signaling pathway starting from a plasma mem- brane α7 nAChR, there is an intracellular pathway engaging the nAChRs expressed in mitochondria outer membrane. This is the first example of func- tional nAChRs expressed in the intracellular or- ganelles. Possibly, it belongs to the most ancient survival mechanisms inherited by mitochondria from their hypothetic prokaryotic ancestor. This novel finding raises a lot of questions concerning the mechanisms of the nAChR targeting to and functioning in mitochondria that is the subject of ongoing studies.
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Structure Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators

Structure Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators

+(! mixture, which was subsequently submerged into the cooling unit containing 100% isopropanol at a temperature of -4°C to -6°C. Depending on flow rates, the buffer entered the oocyte chamber in the range of 13-16°C. Drug plates were kept on ice during washing steps to ensure an application temperature less than 15°C. These were more variable and ranged from 9-14°C, but the main goal of sub-15°C was consistently achieved. Temperature was monitored through a temperature-sensing adaptor for a multimeter (Fluke 116 HVAC Multimeter). Reduction of the experimental temperature can affect the two main sources of variability in these measurements: (1) the extremely fast desensitization of the receptor and (2) the steady increase in peak current over application of a constant concentration of agonist. Since the rates of conformational conversions are correlated with temperature, performing these experiments in a chilled buffer solution can slow conformational rearrangements and reduce some of the variability in the electrophysiological measurements. The steady signal increase can be attributed to the release of more vesicles containing receptors to the surface, which can
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Muscarinic and GABA A receptors modulate acetylcholine release in feline basal forebrain

Muscarinic and GABA A receptors modulate acetylcholine release in feline basal forebrain

Given that most HPLC/EC systems currently used to quantify ACh during relatively short sampling periods cannot detect ACh in brain dialysis samples without the presence of an acetylcholinesterase inhibitor, it has been necessary to work around this limitation. Many in vivo studies have demonstrated that autoreceptors are functional even when a cholinesterase inhibitor is used for dialysis. For example, in the presence of neostigmine, muscarinic antagonists cause concen- tration dependent increases in ACh release in cat pontine reticular formation (Roth et al., 1996;Baghdoyan et al., 1998), rat striatum (Billard et al., 1995), hippocampus (Moor et al., 1995;Kitaichi et al., 1999) and medial septal area (Moor et al., 1995), and mouse prefrontal cortex (Douglas et al., 2001). In cat pontine reticular formation (Baghdoyan et al., 1998) and in rat hippocampus and medial septal area (Moor et al., 1995), muscarinic autoreceptors respond to agonist stimulation by decreasing ACh release. Thus, in the presence of elevated ACh levels due to neostigmine, muscarinic autoreceptors are not maximally inhibited and respond, as predicted, by increasing ACh release in the presence of antagonists and by decreasing ACh release in the presence of agonists.
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Synthesis of Novel Allosteric Agonists and Allosteric Modulators for Nicotinic Acetylcholine Receptors

Synthesis of Novel Allosteric Agonists and Allosteric Modulators for Nicotinic Acetylcholine Receptors

Figure 23 Bar graph of TQS compounds with type II PAM responses at the α7 nAChR 71 Although it is possible that the differences in size between fluorine and the other three halogens could explain the differences in agonist activity observed, differences in chemical properties may also be significant. For example, halogens differ in their electrostatic surface potential and eletronegativities. Indeed, such differences have been suggested to be responsible for the ability of organic compounds containing chlorine, bromine, or iodine (but not those containing fluorine) to form halogen bonds. 74, 75 However, the fact that agonist activity was seen when the halogen atom was replaced by a methyl group 38b or a trifluoromethyl group 36b but not when it was replaced with a hydrogen atom 30b or a hydroxyl group 33b suggests that the sterics and not electronics of the group attached to the 4-position of the phenyl ring
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Early life stress, nicotinic acetylcholine receptors and alcohol use disorders

Early life stress, nicotinic acetylcholine receptors and alcohol use disorders

While circumstantial, this evidence suggests that α4*nAChRs could be an important link between AUDs and stress. Interestingly, human studies have found an association between a family history of alcoholism and: left NAc volume in adolescent females [62]; resting state connectivity of the NAc [128]; and NAc connectivity during reward [129,130] suggesting these individuals have less segregation between the NAc and executive functioning brain regions (like the PFC), and less integration with reward-related brain areas (like the amygdala and VTA). As previously discussed all these brain regions are innervated by cholinergic neurons and contain nAChRs with α4 subunits. Changes in NAc activity were also found following recent negative life stress in individuals with major depressive disorder [38]. Furthermore, polymorphisms in the CHRNA4 gene have been linked to major depression [122] and negative emotionality [131]. While it seems highly likely that the NAc modulates stress-driven alcohol consumption and relapse via α4*nAChRs, it is however difficult to determine whether the α4 subunit is acting alone or in combination with the β4 subunit as the studies discussed above do not explore this possibility. This may be due to the technical difficulties involved separating the functional properties of the individual subunits and the fact that both subunits tend to be expressed together within the brain. Recent advances in transgenic technology utilizing fluorescent tags attached to the various nAChR subunits have the potential for isolating the roles of the individual subunits in this process.
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Gold nanoparticle–choline complexes can block nicotinic acetylcholine receptors

Gold nanoparticle–choline complexes can block nicotinic acetylcholine receptors

Abstract: We identified a novel class of direct ion-channel blockers of ligand-gated ion channels called the gold nanoparticle–choline complex. Negatively charged gold nanoparticles (1.4 nm) block ion pores by binding to the sulfur group of the cysteine loop of nicotinic acetylcholine receptors (nAChRs), and currents evoked by acetylcholine (Ach) can break these bonds. The cur- rent evoked by ACh in nAChRs was blocked directly in ion pores by the gold nanoparticle–choline complex. In adrenal-gland perfusion studies, the complex also blocked nAChRs by diminishing catecholamine release by about 75%. An in vivo study showed muscle relaxation in rats after injection of the complex. These results will foster the application of gold nanoparticles as a direct ion-channel blocker.
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Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

recovery from blocking by PhTX-343 may be caused by its deep binding site in the pore where it may become ‘entrapped’ during channel closure. From previous studies, the mechanism of blocking of nAChR inward currents by PhTX analogues has been proposed to be non-competitive. Bixel et al. 7 showed that α -bungarotoxin did not reduce the binding affinity of N 3 -Ph-PhTX-343-Lys to nAChRs of Torpedo californica, and even a slight increase was noticed with carba- moylcholine. Likewise, inhibition of human muscle-type nAChRs by PhTX-343 and PhTX-12 was not influ- enced by the ACh concentration 8 . Here, our finding that late current inhibition by PhTX-343 and PhTX-12 was not surmountable by increasing the ACh concentration further supports this observation. However, the weaker inhibition of peak current was reduced at high ACh concentrations for PhTX-343 at α 7 and α 1β 1γ δ as well as for PhTX-12 at both tested receptors (i.e. α 3β 4 and α 1β 1γ δ ), suggesting that there may be an element of weak competitive inhibition occurring before the stronger non-competitive use-dependent component masks it. An alternative and perhaps more likely explanation is that slower binding of PhTX as compared to that of ACh means that the faster peak current at high ACh concentrations will be much less affected by PhTX.
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