• International Journal of Oral Biology
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• 제44권2호
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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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• 제50권2호
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• pp.127-131
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• 2007

일련의 새로운 3-benzylidenemyosmine 유도체들의 구조 변화와 미국 바퀴벌래(Periplaneta. americana L.)의 nicotin acetylcholine 수용체 (nAChRs) 사이의 결합 친화력 상수에 관한 정량적인 구조와 활성과의 관계를 분자 홀로그램(H) QSAR 방법으로 검토하였다. 친화력 상수에 관하여 가장 양호한 HQSAR 모델은 분자조각 크기 5${\sim}$8 bin 조건에서 유도된 모델(IV-2)이었다. HQSAR 모델(VI-2)은 높은 예측성(q$^2$=0.507)과 상관성(r$^2_{nev.}$=0.944)에 근거하여 양호한 통계값들을 나타내었다. 그리고 HQSAR 기여도로부터 결합 친화력 상수는 분자내 anabaseine 고리에 의존적이었으며 결합 친화력성이 높은 화합물들이 최적화된 모델(VI-2)에 의하여 설계되었다.

• BMB Reports
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• 제45권5호
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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.

• 대한의생명과학회지
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• 제13권2호
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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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• 제8권4호
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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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• 제13권1호
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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.

• 대한핵의학회지
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• 제36권4호
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• pp.261-270
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• 2002

목적: nAChRs의 ${\alpha}_4{\beta}_2$ 아형에 높은 친화력과 선택성을 갖는 리간드인 $2-[^{18}F]fluoro-A85380([^{18}F]1)$의 효율적인 합성과정을 연구하여 방사화학적 수율, 비방사능, 합성시간, 마우스에서 생물학적 평가의 측면에서 문헌에 보고된 결과들과 비교 평가하였다. 대상 밀 방법: 전구물질의 I을 $^{18}F$으로 치환한 다음 중간물질을 분리하지 않고 trifluoroacetic acid를 가하여 보호기를 제거하였다. 반응혼합액을 HPLC로 정제하여 얻은 방사성리간드($[^{18}F]1$)를 마우스의 꼬리 정맥에 주사한 후에 정해진 시간별로 6개의 뇌조직 (소뇌, 해마, 선조체, 피질, 시상, 상구)을 분리하여 무게를 측정하고 감마계수기를 이용하여 방사능을 측정하였다. 또한 스코폴라민, 메카밀아민, 사이티신, (-)니코틴, 비방사성 표준물질(1)을 마우스에 피하주사로 투여하고 방사성리간드 주사 후 90분에 6개의 뇌조직을 분리하여 무게 및 방사능을 측정하여 %ID/g으로 나타내었다. 이 결과를 대조군과 비교하였다. 결과: $^{18}F$ 표지 후 중간물질을 분리하지 않고 한 번의 HPLC 정제를 통하여 $[^{18}F]1$을 15-20%의 방사화학적 수율로 합성하였으며 비방사능은 $38-55GBq/{\mu}mol$이었다. 이 방사성리간드의 마우스 뇌에서의 분포결과, 주사 후 30분에 nAChRs가 풍부하게 분포되어 있는 시상과 상구에서 가장 많은 섭취를 보였고 이 수용체가 부족한 소뇌에는 거의 섭취되지 않았다. 여러 리간드를 사용하여 $[^{18}F]1$ 섭취의 억제효과를 본 실험에서는 nAChRs 작용제인 사이티신 및 (-)니코틴의 전처리가 시상 및 상구에서 $[^{18}F]1$의 섭취를 70-90% 감소시켰으며, 소뇌에서는 억제효과가 관측되지 않았다. 결론: 이 연구에서 사용된 방법으로 얻어진 $[^{18}F]1$의 방사화학적 수율, 비방사능, 뇌에서의 분포 그리고 여러 리간드에 의한 억제효과는 문헌에 보고된 결과와 유사하였다. 따라서 중간물질을 분리하지 않고 한번의 HPLC를 사용하는 방법이 $[^{18}F]1$의 상용생산에 유용하게 사용될 수 있을 것으로 기대한다.

• Animal cells and systems
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• 제3권2호
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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.

• 한국수산과학회지
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• 제48권1호
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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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• 제27권5호
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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.