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Volume 10 Issue 4
Jul.  2019
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Article Navigation >  Journal of Tropical Biology  > 2019  >  10(4): 307-313

ZHANG Lu, ZHU Xiaopeng, ZHANGSUN Dongting, LUO Sulan. Construction of Nicotinic Acethlcholine Receptor β4 subunit Mutant[J]. Journal of Tropical Biology, 2019, 10(4): 307-313. doi: 10.15886/j.cnki.rdswxb.2019.04.001
Citation: ZHANG Lu, ZHU Xiaopeng, ZHANGSUN Dongting, LUO Sulan. Construction of Nicotinic Acethlcholine Receptor β4 subunit Mutant[J]. Journal of Tropical Biology, 2019, 10(4): 307-313. doi: 10.15886/j.cnki.rdswxb.2019.04.001
shu

Construction of Nicotinic Acethlcholine Receptor β4 subunit Mutant

doi: 10.15886/j.cnki.rdswxb.2019.04.001

  • College of Marine, Hainan University/Ministry of Education Key Laboratory for Tropical Biological Resources/Hiakou Key Laboratory for Marine Drugs, Haikou, Hainan 570228, China

  • Received Date: 2019-03-25
  • Rev Recd Date: 2019-04-29
  • To explain the molecular mechanisms of interaction between β4 nAChR subunit and ligand（agonist ACh and inhibitor α-CTx RegIIA）, a mutant of β4 nAChR subunit was constructed by substituting 168 th Ile（I） with Ser（S） in the Loop F of N-terminal ligand-binding region, by using the method of site-directed mutagenesis that PCR mediated. The results showed that the inward current of oocytes injected with cRNA of α3β4[S168 I] nAChR could be recorded by two-electrode voltage clamp. The mutant receptor had 1.4 times lower sensitivity to α-CTx RegIIA with a higher IC50 of 170 nmol·L-1, compared with the wild-type α3β4 nAChR.
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    [2] WONNACOTT S.Presynaptic nicotinic ACh receptors[J].Trends in Neurosciences,1997,20(2):92-98.
    [3] COLLO G,CAVALLERI L,ZOLI M,et al.Alpha6-containing nicotinic acetylcholine receptors mediate nicotine-induced Structural plasticity in mouse and human iPSC-derived dopaminergic neurons[J].Frontiers in Pharmacology,2018(9):15.
    [4] LIU Q,TANG Z,GAN Y,et al.Genetic deficiency of beta2-containing nicotinic receptors attenuates brain injury in ischemic stroke[J].Neuroscience,2014,256:170-177.
    [5] LUO S,AKONDI K B,ZHANGSUN D,et al.Atypical alpha-conotoxin LtIA from Conus litteratus targets a novel microsite of the alpha3beta2 nicotinic receptor[J].The Journal of Biological Chemistry,2010,285(16):12355-12366.
    [6] NI R Q,MARUTLE A and NORDBERG A.Modulation of alpha 7 nicotinic acetylcholine receptor and fibrillar amyloid-beta interactions in Alzheimer’s disease brain[J].Journal of Alzheimers Disease Jad,2013,33(3):841-851.
    [7] SCHULZ K M,ANDRUD K M,BURKE M B,et al.The effects of prenatal stress on alpha4 beta2 and alpha7 hippocampal nicotinic acetylcholine receptor levels in adult offspring[J].Developmental Neurobiology,2013,73(11):806-814.
    [8] STOKER A K,MARKS M J,MARKOU A.Null mutation of the beta2 nicotinic acetylcholine receptor subunit attenuates nicotine withdrawal-induced anhedonia in mice[J].Europe Journal Pharmacology,2015,753:146-150.
    [9] FUSCO M D,BECCHETTI A,PATRIGNANI A,et al.The nicotinic receptor β2 subunit is mutant in nocturnal frontal lobe epilepsy[J].Nature Genetics,2000,26(3):275-276.
    [10] ZHANGSUN D,ZHU X,WU Y,et al.Key residues in the nicotinic acetylcholine receptor beta2 subunit contribute to alpha-conotoxin LvIA binding[J].Journal of Biological Chemistry,2015,290(15):9855-9862.
    [11] CUNY H,KOMPELLA S N,TAE H S,et al.Key structural determinants in the agonist binding loops of human beta2 and beta4 nicotinic acetylcholine receptor subunits contribute to alpha3beta4 subtype selectivity of alpha-conotoxins[J].Journal of Biological Chemistry,2016,291(45):23779-23792.
    [12] DAWSON A,MILES M F and DAMAJ M I.The beta2 nicotinic acetylcholine receptor subunit differentially influences ethanol behavioral effects in the mouse[J].Alcohol,2013,47(2):85-94.
    [13] DEZFULI G,KELLAR K J,DRETCHEN K L,et al.Evidence for the role of beta2* nAChR desensitization in regulating body weight in obese mice[J].Neuropharmacology,2016,110:165-174.
    [14] 孙志华,罗素兰.烟碱型乙酰胆碱受体及其相关疾病[J].神经药理学报,2017,7(1):53-64.
    [15] JENSEN A A,FROLUND B,LILJEFORS T,et al.Neuronal nicotinic acetylcholine receptors:structural revelations,target identifications,and therapeutic inspirations[J].Journal of medicinal chemistry,2005,48(15):4705-4745.
    [16] 吴勇,朱晓鹏,安婷婷,等.α-芋螺毒素与烟碱型乙酰胆碱受体作用的药理学[J].热带生物学报,2010,1(1):78-84.
    [17] YUAN M,MALAGON A M,YASUDA D,et al.The α3β4 nAChR partial agonist AT-1001 attenuates stress-induced reinstatement of nicotine seeking in a rat model of relapse and induces minimal withdrawal in dependent rats[J].Behavioural Brain Research,2017,333:251.
    [18] LINDOVSKY J,KANIAKOVA M,SVOBODOVA L,et al.Role of negatively charged amino acids in beta 4 F-loop in activation and desensitization of alpha 3 beta 4 rat neuronal nicotinic receptors[J].Biochimica et biophysica acta,2008,1778(4):864-871.
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Construction of Nicotinic Acethlcholine Receptor β4 subunit Mutant

doi: 10.15886/j.cnki.rdswxb.2019.04.001
  • College of Marine, Hainan University/Ministry of Education Key Laboratory for Tropical Biological Resources/Hiakou Key Laboratory for Marine Drugs, Haikou, Hainan 570228, China
  • Received Date: 2019-03-25
  • Rev Recd Date: 2019-04-29

Abstract: To explain the molecular mechanisms of interaction between β4 nAChR subunit and ligand（agonist ACh and inhibitor α-CTx RegIIA）, a mutant of β4 nAChR subunit was constructed by substituting 168 th Ile（I） with Ser（S） in the Loop F of N-terminal ligand-binding region, by using the method of site-directed mutagenesis that PCR mediated. The results showed that the inward current of oocytes injected with cRNA of α3β4[S168 I] nAChR could be recorded by two-electrode voltage clamp. The mutant receptor had 1.4 times lower sensitivity to α-CTx RegIIA with a higher IC50 of 170 nmol·L-1, compared with the wild-type α3β4 nAChR.

ZHANG Lu, ZHU Xiaopeng, ZHANGSUN Dongting, LUO Sulan. Construction of Nicotinic Acethlcholine Receptor β4 subunit Mutant[J]. Journal of Tropical Biology, 2019, 10(4): 307-313. doi: 10.15886/j.cnki.rdswxb.2019.04.001
Citation: ZHANG Lu, ZHU Xiaopeng, ZHANGSUN Dongting, LUO Sulan. Construction of Nicotinic Acethlcholine Receptor β4 subunit Mutant[J]. Journal of Tropical Biology, 2019, 10(4): 307-313. doi: 10.15886/j.cnki.rdswxb.2019.04.001
shu

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