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.
incorporated amide-to-ester backbone mutations into loop B to probe proposed hydrogen bonds to this region. We conclude that the significantly disruptive D89N mutation affects receptor function in several ways: a distortion of the hydrogen bonding network, the introduction of an electrostatic clash between the asparagine amide side chain and the backbone amides of loop B, and, to a lesser extent, a neutralization of charge. We also find that, of the network of hydrogen bonds implied by the AChBP structure (Figure 2.3a), no one hydrogen bond is singularly important. Rather, maintaining the overall network of hydrogen bonds and avoiding electrostatic and/or steric clashes are essential for proper function. To support our unnatural amino acid mutagenesis studies, we conducted molecular dynamics simulations of the wild-type and D89N mutant ligand- binding domains in an effort to evaluate the hydrogen bonding network.
28. Sine, S.M. et al. Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating. J Gen Physiol 120, 483-96 (2002). 29. Xie, Y. & Cohen, J.B. Contributions of Torpedo nicotinicacetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function. J Biol Chem 276, 2417-26 (2001).
62. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Laham, A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A., Gaussian 03, Revision C.02. In 2003. 63. Gleitsman, K. R., Chemical-scalestudies of the nicotinicacetylcholine receptor:
from myasthenia gravis, autoantibodies to the alpha-1 sub- unit of the nicotinicacetylcholine receptor in muscles were shown to disturb neuromuscular signal transduction and mark the cells for complement mediated lysis . Interestingly, also in patients with M. Sjögren, an autoim- mune disease quite often being associated with PBC [24,25], autoantibodies to human muscarinicacetylcholinereceptors (hmAchR) of the M3 type were suggested to be one factor responsible for disease induction [26,27]. More- over, since this specific receptor subtype was also detected on biliary cells but not on hepatocytes [28,29] we hypothe- sized that hmAchR M3-specific autoantibodies could play an important role in the etiopathogenesis of PBC. Thus, we now have undertaken a comprehensive study analyzing whether autoantibodies to the hmAchR of the M3 type could also be found in patients with PBC.
Important progress has been made toward our goal of expressing unnaturals in mammalian cells. The first demonstration of unnatural expression was in the NicotinicAcetylcholine receptor (5). This was an exciting and promising development, but the NMDAR was a more appropriate choice for the project. Earlier work by Sarah Monahan (5) demonstrated wild-type recovery nonsense suppression of the NMDAR channels in mammalian cells. Another study used a serine amber suppressor tRNA to recover wild- type function in a hERG channel (19). Such accomplishments demonstrated that we are able to use this methodology in mammalian cells. What remained was to expand the method to include more cell types, and receptors, and to make the procedures more efficient and robust.
The α7 nicotinicacetylcholine receptor shows broad pharmacology, complicating the development of subtype-specific nicotinic receptor agonists. Here we use unnatural amino acid mutagenesis to characterize binding to α7 by the smoking cessation drug varenicline (Chantix), an α4β2-targeted agonist that shows full efficacy and modest potency at the α7 receptor. We find that unlike binding to its target receptor, varenicline does not form a cation-π interaction with TrpB, further supporting a unique binding mode for the cationic amine of nicotinic agonists at the α7 receptor. We also evaluate binding to the complementary face of the receptor’s binding site by varenicline, the endogenous agonist acetylcholine, and the potent nicotine analog epibatidine. Interestingly, we find no evidence for functionally significant interactions involving backbone NH and CO groups thought to bind the canonical agonist hydrogen bond acceptor of the nicotinic pharmacophore, perhaps reflecting a lesser importance of this pharmacophore element for α7 binding. We also show that the Trp55 and Leu119 side chains of the binding site’s
dramatically reduce channel activity. It remains possible, however, that some combination of hydrogen bonds in this network is important for proper channel function. In addition, this study probed the importance of the α D89 side chain on nAChR function. Mutations that altered side chain length and charge were incorporated at α D89: α D89E, α D89N, and α D89Nha. The α D89E mutation, introducing an extended side chain, resulted in a modest increase in ACh and epibatidine activity, and near wild-type activity for nicotine. Thus, the activity of nicotine tolerates the increased sterics of the α D89E side chain, while the activity of ACh and epibatidine is slightly impaired with this mutation. The α D89N mutation converts the negatively charged wild-type residue to a neutral residue at this position. The α D89N mutation also introduces an electrostatic clash between the amide side chain of α D89N and the backbone amide NHs surrounding α W149. Observations from the present work and previous studies by Sine and co- workers 12 revealed dramatically impaired nAChR receptors in the presence of an α D89N mutation. To further understand the impaired activity of the α D89N mutant, the present study addressed this issue in two ways.
At the time of first publication, [ 18 F]NS14490 showed so far the highest α7 nAChR affinity (2.5 nM) among the published 1,4-diazabicyclo[3.2.2]nonane derivatives in addition with 40- and 400-fold selectivity towards human α3β4 and α4β2 nAChR, respectively [14,22]. Accordingly, an automated radiosynthesis has been developed for fur- ther preclinical PET studies in pigs. After the described optimisation, the labelling yield of the automated radio- synthesis (73% ± 9%; n = 7) as determined with radio-TLC was identical to that of the manual synthesis (71% ± 9%; n = 12) . The radiochemical yield (35% ± 4%) was comparable to that for the previously published radio- chemical automatic synthesis of [ 18 F]fluspidine  the module parameters of which were used as template for the current procedure.
yielded numerous agonists with some therapeutic success (40). As an alternative to selective agonist design, research towards the development and understanding of allosteric modulators – which have the potential to be more target specific and thus produce fewer side effects – has generated progress (32,41). In this study, the use of non-canonical amino acids allowed individual chemical interactions of the agonist binding to the protein to be probed in the presence of the " 7-specific positive allosteric modulator PNU-120596. The conclusion from this analysis is that PNU-120596 does not alter the agonist binding pocket. To further probe the molecular basis of the properties of PNU-120596, conventional mutagenesis throughout the receptor was performed. Several gating interface residues as well as transmembrane residues were identified as vital for propagating PNU-120596 properties throughout the receptor. This network of residues links the agonist binding site to the PNU-120596 binding site and to the channel gate in the pore of the receptor, influencing the global stabilization of the gating equilibrium.
This result is matched with the study on Polish population, which demonstrated a correlation between cigarette smoking per day >10 and allele A of rs1051730 . and the study of Diljit et al who confirmed that smokers carry homozygous mutant genotype for nicotinicacetylcholine receptor inhale more often than others genotypes . Also, Kaur-Knudsen et al found that a variant allele of CHRNA3 is more nicotine dependent .
slow hyperpolarization and a delayed depolarization. The fast depolarization is mediated by receptors that desensitize very rapidly (Szczupak et al. 1993) and are therefore unlikely to respond to basal levels of ACh. The present results indicate that the cholinergic tonus activated the receptors mediating the slow hyperpolarization (Szczupak et al. 1993) and the delayed depolarization (Szczupak et al. 1998), exerting a concomitant depolarizing and hyperpolarizing action on the Rz(X) neurons. Thus, curare, which inhibits both phases of the ACh response, did not produce any net effect by itself. Selective blockers of the delayed depolarization, atropine and strychnine, which also inhibited the neostigmine-induced depolarization, caused a marked hyperpolarization of Rz(X) neurons, shifting their membrane potential to approximately the reversal potential of the Cl − -mediated hyperpolarization (−65 mV) induced by ACh in these neurons (Szczupak et al. 1993). This hyperpolarization did not take place in the presence of curare. Taken together, these results suggest that, under control conditions, the counteracting effects of ACh balanced each other out but that, under conditions of high basal levels of ACh, the depolarizing effect predominated. As expected, Retzius neurons from the reproductive segments were not affected by atropine or strychnine, since the expression of the receptors mediating the long-lasting depolarization is very low (Szczupak et al. 1998) and they did not therefore depolarize when AChE activity was inhibited.
on cholinergic nerve terminals. Blockade of the M2 autoreceptor will cause increased release of acetylcholine , which could cause competition between ACh and [ 11 C]AF150(S) for the M1ACh-R, located postsynaptically. In microdialysis studies in rats, AF-DX 384 given IP at a dose of 5 mg kg −1 resulted in a long-lasting significant in- crease in acetylcholine release, 30 min after administra- tion, in both the cortex and hippocampus . In those studies, breakdown of extracellular ACh was prevented by adding the AChE inhibitor physostigmine in the microdi- alysis perfusate. In the present study, AChE inhibition as protection against ACh breakdown was attempted by using co-treatment with rivastigmine . AF-DX 384 and rivastigmine, at the present dose, did not cause animal immobility or catalepsy, and such effect on behaviour by AF-DX 384 treatment has neither been reported in the lit- erature. Therefore, the effect-size of M2ACh-R blockade on acetylcholine release probably is much less pronounced than that of haloperidol. Pre-treatment with only AF-DX 384 also did not have an effect on BP ND in M1ACh-R-rich
Phosphorylation of nAChRs has been showed to modulate receptor activity in the nematode Ascaris suum. We expect that protein kinases also modulate levamisole receptors in C. elegans. For further investigation of this hypothesis, we can either apply protein kinase catalytic subunits and ATP to test channel activity, or use some genetic manipulated mutants such as TAX-6 to investigate effects of phosphorylation. Another future study interesting to us is that the role of nicotine sensitive nAChRs. This nAChR subtype has been showed to contribute ~80% of the whole-cell current induced by acetylcholine, but the knockout of this nAChR has no observable phenotype. The nicotine sensitive receptor and levamisole sensitive receptor may have different Ca 2+ permeability or induce different Ca 2+ cascade. To investigate the Ca 2+ permeability of nicotine sensitive nAChRs, I propose to use Ca 2+
A difficulty in assessing the field relevance of studies is that laboratory studies often do not provide a natu- ral choice of forage and so the dose of pesticide delivered may exceed that encountered in the environment. In contrast, field studies limit exposure of bees to the field conditions under study (usually the flowering period of a crop) to minimize exposure to unknown confounding factors such as other pesticides. However, this also creates an artificial situation, with bees on site for a short duration. This has 2 problems. Firstly, a prolonged neonicoti- noid exposure by its translocation to wildflowers 8,10–12 is excluded from the study. Secondly, the expected deficit
Previous results from our laboratory have shownpharmacological activation of nicotinicacetylcholinereceptors (nAChRs) to produce a sustained pro-epileptogenic effect within the hippocampal slice (Roshan-milani et al 2003). This was observed across a range of different forms of pharmacologically-induced epileptiform activity, including 4-aminopyridine (4AP), bicucullineand low magnesium models, suggesting that nAChRs may have a general action in exacerbating epilepsy-like discharges. The mechanisms underlying the pro-epileptogenic action of nAChRs are complex and not simply due to the activation of nAChRs, and a number of factors may contribute to their pro-epileptogenic actions. An important locus of action is likely to be GABAergic circuits. nAChRs can have a potent impact upon the activity of interneurone populations which themselves are important regulators of network excitability. Thus, it is likely that the pro-epileptogenic action of nAChR activation is mediated in part through GABAergic circuits.However, such an action of nAChRs appears to be independent of fast GABAergic transmission since the pro-epileptogenic actions of nAChR activation is resistant to the blockade of GABA A receptors (Roshan-
In addition to Lynx1 expression on PV + interneurons, we find that a separate member of the Lynx family, Lypd6 is enriched in a population of deep layer SST in- terneurons without expression on any other GABAergic subtype. Most cortical SST interneurons are Martinotti cells which send their axons into superficial layers where they form dense axon collateral networks in layer I . Little is known about the nicotinic responsiveness of specific cortical SST subpopulations, however, Martinotti cells are morphologically and physiologically similar to oriens-lacunosum moleculare (O-LM) neurons of the hippocampus  that have been shown to be highly responsive to nicotinic signaling [60,61] and also densely express Lypd6 (Additional file 1: Figure S5). Nicotinic activation of O-LM neurons in CA1 facilitates long term potentiation through increases in calcium influx [61,62]. The activation or potentiation of O-LM neurons as well as neurons in the central amygdala is required for cer- tain forms of experience dependent fear learning [63,64]. Lypd6 is an ideal candidate to mediate or modulate these activities through its ability to directly potentiate calcium currents through nicotinicreceptors . In cortical circuits, little is known about the involvement of SST neurons in experience-dependent plasticity but their positioning within cortical circuits make them an intri- guing candidate to govern OD plasticity where the ex- pression of Lypd6 could modulate any impact that they have. SST neurons highly innervate local PV cells placing them in an ideal position to drive the inhibition instructive to plasticity [48,65-67]. Additionally, SST neurons may dir- ectly mediate plasticity mechanisms through the inhibition of incoming information to the distal dendrites of pyr- amidal neurons [68,69]. These observations highlight the importance of future work designed to elucidate the role of SST neurons in OD plasticity and how these functions may be mediated by molecular modulators such as Lypd6.
axons and project to the brainstem or the thalamocortical system.  The impulses originated from brain stem nuclei, “descend” to the spinal level and affect the transmission of pain signals at the DH. [3,4] The relative balance between descending inhibition and facilitation can be changed by the type and intensity of the stimulus and also by the time following the injury. Somatosensory system that detects destructive and potentially tissue injurious stimuli plays a critical role as an essential protective mechanism including numerous interacting peripheral and central mechanisms.  These mechanisms are the highly complex process involving various mediators and receptors as seen in Table 1. Pain control is provided by the interaction of these chemicals and receptors over an extensive network from the periphery to the CNS. The rate of participation of the chemicals and receptor types in the modulation depend on the pain types and noxious stimulus.
Although, a1nAchRs were identified in the areas of PNL infiltration, even with higher magnification it was difficult to identify the exact cell type expressing the IR. As the abscess site attracted a large amount of macro-phages and PNL, we could assume that a1nAChRs are expressed in the live cells of the peripheral rim of the abscess. This suggests that a1 subtype of nicotinicreceptors may play a role in the anti-inflammatory response in localised inflammation. The subcutaneous lymph node associated with the inflammatory region also expressed a1nAChR IR in its capsular, subcapsular sinuses and trabeculae regions. This is also suggestive of anticho-linergic control of inflammation.