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Nicotinic acetylcholine receptors(nAChRs) is one of the main receptor of acetylcholine in the body. It is an excitatory transmembrane cation channel. It is confirmed that nAChRs has important physiological functions in both the nervous system and non-nervous system, and is related to many diseases.nAChRs has also been confirmed to be related to many diseases, so its structure And functional research is very necessary. Therefore, it is necessary to study the structure and function of nAChRs. It is known that the intracellular loop of the nicotinic receptor protein plays a major role in regulating its intracellular metabolism and downstream signal transduction. However, the structure and function of this protein sequence are still unclear. In this paper, the current research status of nicotinic receptor intracellular interaction proteins were reviewed, aiming to further explore the structural and functional characteristics of nicotinic receptor intracellular loops through the information of their interaction proteins and interaction sites, and to provide ideas for clinical targeted therapy.
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Abstract Galanthamine is an Amaryllidaceae-derived acetylcholinesterase inhibitor used to treat memory impairment in Alzheimer's disease and vascular dementia. There is evidence that galanthamine, in addition to its effects on acetylcholinesterase, may enhance or inhibit brain nicotinic acetylcholine receptors, which could increase or decrease the therapeutic efficacy of galanthamine, respectively. Here, we evaluated the effects of galanthamine and two others Amaryllidaceae acetylcholinesterase inhibitors (haemanthamine and tazettine) analyzed by gas chromatography-mass spectrometry and identified by comparing their mass fragmentation patterns with literature and database NIST vs.2.0 on the agonist responses of brain nicotinic acetylcholine receptors α7, α3β4, (α4)2(β2)3 and (α4)3(β2)2. Using nicotinic acetylcholine receptors expressed heterologously in Xenopus oocytes, in conjunction with two-electrode voltage clamping, we found that galanthamine inhibits the function of nicotinic acetylcholine receptors assayed through a mix competitive and non-competitevely. Nicotinic acetylcholine receptor α7 were significantly more sensitive to inhibition (17 ± 0.6 µM) than the heteromeric receptor, α3β4 (90 ± 3.4 µM). Neither haemanthamine nor tazettine were more potent than galanthamine.
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<p><b>OBJECTIVE</b>To explore the role of β2-nicotinic acetylcholine receptor (β2-nAChR) in the development of γ- aminobutyric acid A type receptors (GABA-Rs) in hippocampal CA1 and CA3 pyramidal neurons of mice.</p><p><b>METHODS</b>The hippocampal CA1 and CA3 pyramidal neurons were acutely isolated from β2-nAChR gene knockout (β2-KO group) mice. GABA currents in CA1 and CA3 pyramidal neurons were induced with the selective GABA-R agonist muscimol and recorded using perforated patch-clamp recording technique. The GABA currents of CA1 and CA3 pyramidal neurons were tested for their equilibrium potentials (Es) and kinetic parameters and were compared with the measurements in wild-type mice (WT group).</p><p><b>RESULTS</b>The mean E of CA1 neurons (=7) of β2-KO mice (=4) was -31.7±3.5 mV, showing an obvious depolarizing shift compared with the WT mice ( < 0.05); the mean E of CA3 neurons (=4) was -16.1±4.6 mV, also showing a depolarizing shift ( < 0.01). The difference in the Es between CA3 and CA1 neurons in β2-KO mice, but not in WT mice, was significant ( < 0.05). The GABA-R desensitization was significantly slowed down in both CA1 and CA3 neurons of β2-KO mice, with decay time of 2.2±0.2 s and 3.2±0.1 s, respectively, significantly longer than those in WT mice (1.6±0.1 s and 2.3±0.1 s, respectively; < 0.05).</p><p><b>CONCLUSIONS</b>β2-containing nAChRs may promote the functional maturation of GABA-R in CA1 and CA3 pyramidal cells in mouse hippocampus.</p>
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Objective To explore the impact of exercise on the learning and memory of rats undergoing nicotine withdrawal and its underlying mechanism.Methods Forty four-week-old male SpragueDawley rats were subjected to nicotine conditioned place preference(CPP) training before being randomly separated into a high-,moderate-,low-intensity exercise and control(no exercise) group.Rats in exercise groups underwent the treadmill running at low,moderate or high intensity 30 minutes per day for 10 days consecutively.The nicotine-associated context memory was evaluated using the CPP preference score.Morris water maze (MWM) tasks were used to examine the spatial learning and memory.The protein level of α7 acetylcholine receptors(nAChRs) in the prefrontal cortex and hippocampus was visualized by Western blotting.Results Rats undergoing exercises at a high or moderate intensity had a significantly lower CPP score than the control group (P＜0.01 and P＜0.05 respectively).However,there was no significant difference in the CPP score between the low-intensity exercise and control groups.Only rats doing moderate-intensity exercise presented a significantly shorter escape latency than controls in the MWM place navigation test(P＜0.01).Significant increase in time spent and distance swam in the target quadrant was observed in the moderate-and high-intensity exercise group,but without significant differences between the two groups.Moreover,a significant increase in the number of crossing target quadrant was only observed in rats that exercised at a moderate intensity (P＜ 0.05).The protein level of α7 nAChRs was significantly elevated in the prefrontal cortex(P＜0.01),but not in the hippocampus of rats doing moderate intensity exercise.Conclusion Ten-day treadmill running at a moderate intensity may improve learning and memory performance,and facilitate the extinction of nicotine reward memory of nicotine-treated rats via increasing α7 nAChR-mediated signaling in the frontal cortex.
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OBJECTIVE: To construct the point mutants of α3* nicotine acetylcholine receptors (nAChRs), optimize the method of receptor mutagenesis and investigate the function of the mutants by using the agonist acetycholine (Ach). METHODS: The α3* nAChRs mutants were constructed by PCR mediated site-directed mutation techniques. Point mutated primers were designed according to rat α3 subunit gene. The cRNA of α3 subunit point mutant was synthesized by in vitro transcription. The expression of mutants in Xenopus oocytes were detected by two-electrode voltage-clamp techniques. Gating properties of the two mutants were detected by Ach. RESULTS: Mutants of α3β2 and α3β4 nAChRs subtypes were constructed successfully. The half effective concentrations (EC50) of wild types α3β2 and α3β4 nAChRs were 55.33 and 163.00 μmol·L-1, respectively. While the EC50 of α3(S147T)β2 and α3(S147T)β4 nAChRs mutants were 33.10 and 121.10 μmol·L-1, respectively. CONCLUSION: The construction of mutation from the 147th serine to threonine of α3 subunit can provide a function model to make more other receptor mutants, and would be helpful to interrogate the interaction between drug and α3* nAChR.
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Polymorphisms in the nicotinic acetylcholine receptor subunit CHRNA5 gene have been associated with lung cancer positive susceptibility in European and American populations. In the present hospital-based, case-control study, we determined whether polymorphism in rs503464 of CHRNA5 is associated with lung cancer risk in Chinese individuals. A single nucleotide polymorphism in CHRNA5 rs503464, c.-166T>A (hereafter T>A), was identified using TaqMan-MGB probes with sequencing via PCR in 600 lung cancer cases and 600 healthy individuals. Genotype frequencies for rs503464 (T>A) were in Hardy-Weinberg equilibrium for the control population. However, genotype frequencies were significantly different between cases and controls (P < 0.05), while allele frequencies were not significantly different between groups. Compared to homozygous genotypes (TT or AA), the risk of lung cancer in those with the heterozygous genotype (TA) was significantly lower (OR = 0.611, 95%CI = 0.486-0.768, P = 0.001). Using genotype AA as a reference, the risk of lung cancer for those with genotype TA was increased 1.5 times (OR = 1.496, 95%CI = 1.120-1.997, P = 0.006). However, no difference in risk was observed between T allele carriers and A allele carriers (OR = 0.914, 95%CI = 0.779-1.073, P = 0.270). Stratification analysis showed that the protective effect of TA was more pronounced in those younger than 60 years, nonsmokers, or those without a family history of cancer, as well as in patients with adenocarcinoma or squamous cell carcinoma in clinical stages III or IV (P < 0.05). Therefore, the heterozygous genotype c.-166T>A at rs503464 of CHRNA5 may be associated with reduced risk of lung cancer, thus representing a susceptibility allele in Chinese individuals.
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Female , Humans , Male , Middle Aged , Lung Neoplasms/genetics , Polymorphism, Genetic/genetics , Receptors, Nicotinic/genetics , Case-Control Studies , China , Gene Frequency , Genetic Predisposition to Disease , Genotype , Lung Neoplasms/pathology , Neoplasm StagingABSTRACT
The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as alpha3beta4, alpha7 and alpha9alpha10. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current (IACh) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited IACh. Pre-treatment of quercetin further inhibited IACh in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of IACh by quercetin was reversible and concentration-dependent. The IC50 of quercetin was 18.9+/-1.2 microM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of IACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.
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Adult , Humans , Acetylcholine , Flavonoids , Fruit , Ginkgo biloba , Inhibitory Concentration 50 , Ligand-Gated Ion Channels , Solanum lycopersicum , Molecular Weight , Neuromuscular Junction , Onions , Oocytes , Quercetin , Receptors, Nicotinic , RNA, Complementary , Vegetables , XenopusABSTRACT
Objective To observe the learning and memory changes in coal-burning type of fluorosis rats, detect the expressions of neuronal nicotinic acetylcholine receptor(nAChR) at mRNA and protein levels in rat brains and to reveal the mechanism of changed learning and memory ability. Methods Twenty-four healthy SD rats, weighting 100 - 120 g, were randomly divided into three groups(8 in each). Control group was fed with normal diet, and low- and high-dose fluoride groups were fed with corn polluted with high fluoride (fluoride were 11.30,104.20 mg/kg, respectively) during drying processes with local burning-coal from the areas of endemic fluorosis to established rat model of chronic fluorosis. After exposed to fluoride for 6 months, behavioral changes were measured by Morris water maze. Animals were sacrificed, the brain was taken, after homogenizing the fluoride content of brain tissue was determined by fluoride ion selective electrode. The α3, α4 and α7 nAChR subunits at mRNA and protein levels were analyzed by real-time PCR and Western blotting, respectively. Results For rats in low- and high-fluoride groups, the escape latency time[(12.42 ± 8.03),(17.48 ± 8.05)s] was significantly longer than that in the control[(7.04 ± 3.29)s, all P< 0.05]. For rats in high-fluoride group, the numbers of crossing the platforms (1.62 ± 0.87) and the time of staying at the platforms[(16.70 ± 5.02)s] were significantly decreased as compared to that of control[3.53 ± 1.67, (23.33 ± 5.35)s, all P < 0.05]. The fluoride content in rat brain tissue in low- or high-fluoride groups [(1.14 ± 0.04), (1.79 ± 0.04)mg/kg] was significantly higher than that of control [ (0.52 ± 0.05) mg/kg, all P < 0.05]; in addition, the amount of fluoride in brain tissue of high-fluoride group was significantly higher than that of low-fluoride group(P < 0.05). In high-fluoride group, the mRNA expressions of α3, α4 and α7 nAChR subunits in rat brains(1.51 ± 0.20,1.45 ± 0.06,1.63 ± 0.08) were significantly lower as compared to controls (1.79 ± 0.11,1.66 ± 0.14,1.83 ± 0.06, all P< 0.05); whereas there were no significant changes in mRNA levels of these receptor subunits of the rat brains between low-fluoride group(1.65 ± 0.17,1.59 ± 0.09,1.71 ± 0.03) and controls (all P > 0.05). Furthermore, the protein levels of α3, α4 and α7 nAChR subunits in rat brains of highfluoride group(0.58 ± 0.13,0.16 ± 0.03,1.41 ± 0.38) and low-fluoride group(0.56 ± 0.23,0.08 ± 0.02,0.51 ± 0.16) were significantly lower than those of controls( 1.48 ± 0.42,0.57 ± 0.21,2.56 ± 0.26, P<0.05 or < 0.01). Conclusions Decreased ability of learning and memory in coal-burning type of fluorosis rats may be associated with declined expressions of nAChR at proteins and mRNA levels, which might be the main mechanism of the behavior change.
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Three dimensional quantitative structure activity relationship between diazabicyclo[4.2.0]octanes and nicotinic acetylcholine receptor (h alpha4beta2 and h alpha3beta4) 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 (q2=0.926 and 0.945, r2(ncv)=0.983 and 0.988). This study can be used to develop potent h alpha4beta2 receptor agonists with low activity on h alpha3beta4 subtype.
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Quantitative Structure-Activity Relationship , Receptors, NicotinicABSTRACT
Objective To explore the effects of stimulating vagus nerve with pulse current on peripheral blood CD4+T lymphocyte of rats with collagen induced arthritis and its mechanism.Methods To duplicate model rats of experimental arthritis(EA)by intradermal injection of Ⅱtype collagen,divide the rats into 2 groups:vagus nerve stimulation(VNS)group and sham operated group.Rats in VNS group were stimulated at the left cervical vagus nerves for 30 minutes a day with constant square wave,pulse current with intra train of 16 Hz,pulse duration of 1.0 ms,train duration of 10 s,interstimulus interval of 1.5 min and intensities of 3.0 mA.Then flow cytometry and immunofluorescence methods were used to detect the activation of CD4+T lymphocytes(expressing CD71)and the expression of nicotinic acetylcholine receptors alpha 7(nAChR?7)and choline acetyltransferase(ChAT)in peripheral blood CD4+T lymphocytes.Results In VNS group,the expression of nAChR?7 and ChAT were significantly raised in CD4+ T cells at 1st weekend(Pa
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Aim To investigate the desensitization characteristics of neuronal ?4?2、?4?4 and ?7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 cell line stimulated by prolonged of repetitive application of agonist.Methods The Whole cell recording configuration of patch-clamp technique was used to study the desensitization characteristics of ?4?2、?4?4 and ?7-nAChRs in cultured SH-EP1 cells three days after passage by rapid application of nicotine onto the soma of the cells.Results Inward currents were elicited by rapid application of agonist in all the three types of SH-EP1 cells in a concentration-dependent manner.?7-nAChRs desensitized the fastest and deepest in the three while ?4?4-nAChRs the slowest and lightest under prolonged stimulation.?4?2-nAChRs desensitized the fastest in the three under repetitive stimulation while ?4?4 and ?7-nAChRs didn't have obvious desensitization.Conclusion When stimulated by prolonged of repetitive application of agonist,neuronal ?4?2、?4?4 and ?7-nicotinic acetylcholine receptors had different desensitization characteristics,which is possibly associated with their roles in organophosphate poisoning inducing persistent stimulation of excessive accumulation of endogenous ACh and in nicotine addiction inducing repetitive stimulation of nicotine.
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Aim To investgate the mechnism through which ginkgolides affect learning and memory capabilities of the Alzheimers disease-like rats. Methods Okadaic acid(OA)was injected into the CA1 region of the rat hippocampus and the rats were gavaged with ginkgolides. The learning and memory abilities of the rats were assessed through Morris water maze behavioral test, and the expressions of nicotinic acetylcholine receptors and ChAT were observed by Western blotting and immunohistochemistry, respectively.Results Compared with the control rats, the capabilities of learning and memory were lowered significantly(P
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Objective To observe the regulation of ?-7 neuronal nicotinic acetylcholine receptors(?-7-nAChRs) by nicotine in the ventral tegmental area(VTA),the substantia nigra(SN) and the cortex of the rat. Methods Nicotine exposed animal models were established.The changes of ?-7-nAChRs proteins and mRNA were observed by immunohistochemistry and in situ hybridization in VTA,SN and cortex,and the expression of ?-7-nAChRs proteins by Western blotting in PC12 cells. Results The expression of ?-7-nAChRs proteins in cortex and mRNA in VTA and SN were upregulated by nicotine.The expressions of ?-7-nAChRs in PC12 cells were in proportion to and dependent on the time for nicotine to function and the dose.Conclusion Both ?-7-nAChRs mRNA in VTA and SN in transcriptional mechanism and ?-7-nAChRs protein in the cortex at the translation level were upregulated.