• International Journal of Oral Biology
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• v.44 no.2
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• pp.50-54
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• 2019

Melatonin is a neurotransmitter that modulates various physiological phenomena including regulation and maintenance of the circadian rhythm. Nicotinic acetylcholine receptors (nAChRs) play an important role in oral functions including orofacial muscle contraction, salivary secretion, and tooth development. However, knowledge regarding physiological crosstalk between melatonin and nAChRs is limited. In the present study, the melatonin-mediated modulation of nAChR functions using bovine adrenal chromaffin cells, a representative model for the study of nAChRs, was investigated. Melatonin inhibited the nicotinic agonist dimethylphenylpiperazinium (DMPP) iodide-induced cytosolic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) increase and norepinephrine secretion in a concentration-dependent manner. The inhibitory effect of melatonin on the DMPP-induced $[Ca^{2+}]_i$ increase was observed when the melatonin treatment was performed simultaneously with DMPP. The results indicate that melatonin inhibits nAChR functions in both peripheral and central nervous systems.

• Applied Biological Chemistry
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• v.50 no.2
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• pp.127-131
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• 2007

The molecular design and holographic (H) quantitative structure-activity relationships (HQSARs) on the binding affinity constants between new 3-benzylidenemyosmine analogues and nicotin acetylcholine receptors (nAChRs) of American cockroach (Periplaneta. americana L.) were studied quantitatively. The optimized HQSAR model (IV-2) for the binding affinity constants was derived from fragment distinction of hydrogene atoms in fragment size, 5${\sim}$8 bin. The statistical results of the HQSAR model (IVI-2) exhibited the best predictability and fitness for the binding affinity constants based on the cross-validated value (q$^2$=0.507) and non cross-validated value (r$^2_{nev.}$=0.944). From the graphical analyses of atomic contribution maps, it was revealed that the binding affinity constants depends upon the anabaseine ring in molecule and the most active compounds were designed by optimized HQSAR model (VI-2).

• BMB Reports
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• v.45 no.5
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• pp.275-280
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• 2012

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of homo- or heteropentameric ligand-gated ion channels. Understanding the physiological role of each nAChR subtype and the key residues responsible for normal and pathological states is important. ${\alpha}$-Conotoxin neuropeptides are highly selective probes capable of discriminating different subtypes of nAChRs. In this study, we performed homology modeling to generate the neuronal ${\alpha}3$, ${\beta}2$ and ${\beta}4$ subunits using the x-ray structure of the ${\alpha}1$ subunit as a template. The structures of the extracellular domains containing ligand binding sites in the ${\alpha}3{\beta}2$ and ${\alpha}3{\beta}4$ nAChR subtypes were constructed using MD simulations and ligand docking processes in their free and ligand-bound states using ${\alpha}$-conotoxin GIC, which exhibited the highest ${\alpha}3{\beta}2$ vs. ${\alpha}3{\beta}4$ discrimination ratio. The results provide a reasonable structural basis for such a discriminatory ability, supporting the idea that the present strategy can be used for future investigations on nAChR-ligand complexes.

• Biomedical Science Letters
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• v.13 no.2
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• pp.119-125
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• 2007

It is widely known that protein tyrosine kinases (PTKs) are involved in controlling many biological processes such as cell growth, differentiation, proliferation, survival and apoptosis. An $\alpha3\beta4$ subunit combination acts as a major functional acetylcholine receptor (nAChRs) in male rat major pelvic ganglion (MPG) neurons, and their activation induces fast inward currents and intracellular calcium increases. Recently it has been reported that the activity of acetylcholine receptors (AChRs) in some neurons can be negatively regulated by PTKs. However, the exact mechanism of regulation of nAChRs by PTKs is poorly understood. Therefore, we examined the potential role particular in nAChR by PTK using electrophysiology and calcium imaging in male rat MPG neurons. ACh induced inward currents and $(Ca^{2+})_i$ increases in MPG neurons, concomitantly. These responses were inhibited by more than 90% in $Na^+$- or $Ca^{2+}$- free solution. $\alpha$-conotoxin AuIB, a selective $\alpha3\beta4$ nAChR blocket, inhibited ACh-induced inward currents. Genistein (10 $\mu$M), a broad-spectrum tyrosine kinase inhibitor, markedly decreased ACh-induced currents and $Ca^{2+}$ transients, whereas 10 $\mu$M genistin, an inactive analogue, had little effect. Overall these data suggest that the activities of $\alpha3\beta4$ AChRs in MPG neurons are positively regulated by PTK. In conclusion, trosine kinase may be one of the key factors in the regulation of $\alpha3\beta4$ nAChRs in rat MPG neurons, which may play an important roles in the autonomic neuronal function such as synaptic transmission, autonomic reflex, and neuronal plasticity.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.4
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• pp.219-225
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• 2004

The pelvic ganglia provide autonomic innervations to the various urogenital organs, such as the urinary bladder, prostate, and penis. It is well established that both sympathetic and parasympathetic synaptic transmissions in autonomic ganglia are mediated mainly by acetylcholine (ACh). Until now, however, the properties of ACh-induced currents and its receptors in pelvic ganglia have not clearly been elucidated. In the present study, biophysical characteristics and molecular nature of nicotinic acetylcholine receptors (nAChRs) were studied in sympathetic and parasympathetic major pelvic ganglion (MPG) neurons. MPG neurons isolated from male rat were enzymatically dissociated, and ionic currents were recorded by using the whole cell variant patch clamp technique. Total RNA from MPG neuron was prepared, and RT-PCR analysis was performed with specific primers for subunits of nAChRs. ACh dose-dependently elicited fast inward currents in both sympathetic and parasympathetic MPG neurons $(EC_{50};\;41.4\;{\mu}M\;and\;64.0\;{\mu}M,\;respectively)$. ACh-induced currents showed a strong inward rectification with a reversal potential near 0 mV in current-voltage relationship. Pharmacologically, mecamylamine as a selective antagonist for ${\alpha}3{\beta}4$ nAChR potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons $(IC_{50};\;0.53\;{\mu}M\;and\;0.22\;{\mu}M,\;respectively)$. Conversely, ${\alpha}-bungarotoxin$, ${\alpha}-methyllycaconitine$, and $dihydro-{\beta}-erythroidine$, which are known as potent and sensitive blockers for ${\alpha}7$ or ${\alpha}4{\beta}2$ nAChRs, below micromolar concentrations showed negligible effect. RT-PCR analysis revealed that ${\alpha}3$ and ${\beta}4$ subunits were predominantly expressed in MPG neurons. We suggest that MPG neurons have nAChRs containing ${\alpha}3$ and ${\beta}4$ subunits, and that their activation induces fast inward currents, possibly mediating the excitatory synaptic transmission in pelvic autonomic ganglia.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.55-59
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• 2009

Three dimensional quantitative structure activity relationship between diazabicyclo[4.2.0]octanes and nicotinic acetylcholine receptor($h{\alpha}4{\beta}2$ and $h{\alpha}3{\beta}4$) agonists was studied using comparative molecular field analysis(CoMFA) and comparative molecular similarity indices analysis(CoMSIA). From 11 CoMFA and CoMSIA models, CoMSIA with steric and electrostatic fields gave the best predictive models($q^2=0.926$ and 0.945, ${r^2}_{ncv}=0.983$ and 0.988). This study can be used to develop potent $h{\alpha}4{\beta}2$ receptor agonists with low activity on $h{\alpha}3{\beta}4$ subtype.

• The Korean Journal of Nuclear Medicine
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• v.36 no.4
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• pp.261-270
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• 2002

Purpose: Nicotinic acetylcholine receptors (nAChRs), which mediate excitatory neurotransmission, are known to participate in various neurophysiological functions. Severe losses of nAChRs have been noted in Alzheimer's and Parkinson's diseases. Therefore, noninvasive and quantitative imaging of nAChRs would offer a better understanding on the function of these receptors. In this study, $2-[^{18}F]fluoro-A85380\;([^{18}F]1)$, an ${\alpha}_4{\beta}_2$ nAChRs radioligand, was prepared using one HPLC purification and evaluated in mouse brain, and the results were compared with those in the literature. Materials and Methods: $[^{18}F]1$ was prepared by $[^{18}F]$fluorination of the iodo precursor followed by acidic deprotection and then purified by HPLC. Tissue distribution studies were performed in mouse brain at the indicated time points and the result was expressed as %ID/g. Inhibition studies were also carried out with pretreatment of various ligands. Results: One HPLC purification method gave the desired product in 15-20% radiochemical yield and with high specific activity ($38-55GBq/{\mu}mol$). Tissue distribution studies showed that $[^{18}F]1$ specifically labeled nAChRs in mouse brain with a high thalamus to cerebellum uptake ratio (13.8 at 90 min). Inhibition studios demonstrated selective binding of $[^{18}F]1$ to nAChRs, blocking the uptake of the $[^{18}F]1$ in nAChR-rich legions by selective ligands such as cytisine and nicotine which are well-known nAChRs agonists. Conclusion: This study demonstrated that the $[^{18}F]1$ produced by the method using one HPLC purification gave the results similar to those reported in the literature. Therefore, this synthetic method can be readily applied to the routine preparation of $[^{18}F]1$, a PET radioligand for ${\alpha}_4{\beta}_2$ nAChRs imaging.

• Animal cells and systems
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• v.3 no.2
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• pp.181-185
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• 1999

Xenopus oocyte is one of the widely used heterologous expression systems of ion channels for electrophysiological studies. Here we describe a new method in which cRNA produced by polymerase chain reaction (PCR) and in vitro transcription is injected to express ion channels in oocytes. This method enables us (1) to eliminate all or a part of the untranslated region of the cDNA and to replace it with a known sequence which helps increase the expression level in oocytes, and (2) to use the PCR product for in vitro transcription without subcloning. Using this method, the expression level of one of the neuronal nicotinic acetylcholine receptors (nAChRs) $\alpha$$_{6}$ subtype in oocytes was systematically increased by more than 100-fold, which was confirmed both by the $\alpha$-Bungarotoxin ($\alpha$,/TEX>Bgt) binding assay and the current measurement.t.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.1
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• pp.71-75
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• 2015

Nicotinic acetylcholine receptors (nAChRs) are essential for neurotransmission and important therapeutic targets of diseases related to neurotransmission. A recent experimental study identified three residues (Lys185, Asp187, and Ile188) of the ${\alpha}6$ nAChR subunit as determinants of ${\alpha}$-conotoxin BuIA selectivity, yet how these residues confer toxin selectivity remains unclear. In this study, we performed all-atom molecular dynamics simulations with two toxin-bound ${\alpha}4{\beta}2$ nAChR systems: the wild-type ${\alpha}4{\beta}2$ and one in which we replaced the three ${\alpha}4$ subunit residues with three ${\alpha}6$ subunit residues identified in an experimental study (Tyr185Lys, Thr187Asp, and Arg188Ile). After mutation, Asp199 lost the salt bridge formed with Arg188 in the wild type located around loop C. Then, the loop C conformation changed and became more flexible than that of the wild type. We also detected reduced space between the toxin and the binding site in the mutant simulation, resulting in increased binding affinity to the toxin. Therefore, we propose a new Asp199 mutation that breaks the salt bridge and may produce similar selectivity to that of the Arg188 mutation.

• Molecules and Cells
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• v.27 no.5
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• pp.591-599
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• 2009

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not ${\alpha}7$, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of ${\alpha}7$ nAChR induces alterations in channel gating properties and converts ${\alpha}7$ nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside $Rg_3$ ($Rg_3$) activity against the ${\alpha}7$ nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to $Rg_3$. We further characterized $Rg_3$ regulation of L247T receptors. We found that $Rg_3$ inhibition of mutant ${\alpha}7$ nAChR channel currents was reversible and concentration-dependent. $Rg_3$ inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between $Rg_3$ and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in $Rg_3$ interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that $Rg_3$ forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas $Rg_3$ localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for $Rg_3$ at the channel pore.