| Nicotine acetylcholine receptor(nAChR)is a kind of ligand-gated ion channel composed of a pentameric structure.The ligand-binding sites are concentrated in the extracellular N-terminal amino acid region of the receptor.According to the distribution of the issue,the classification of nAChRs can be divided into neuronal-type nAChRs and muscle-type nAChRs.According to the composition of subunits,the classification of nAChRs can be divided into homologous nAChRs(a7)and heterologous nAChRs(α3β2,α3β4,α4β2).Neuronal nAChRs are associated with pathophysiological processes such as depression,nicotine addiction,Alzheimer’s disease,which can be important targets for related drugs.α3β2 and α3β4 nAChRs are mainly distributed in the central and peripheral nervous system,with different pathological and physiological functions.α3β2 nAChR is mainly related to pain while α3β4 nAChR is mainly related to nicotine,drug abuse,lung cancer and other diseases.The β2 and β4 subunits are one of the important reasons for the difference in function and structure between the two subtypes.The amino acid sequences of β2 and β4 subunits have high homology,and the extracellular N-terminal domains of Loop D,E,and F are the major regions of ligand binding.Determining the key amino acid sites of β2 subunit ligand-binding region is of great significance for studying the differences in receptor function,screening subtype-specific drugs and studying the molecular mechanism of drug action mechanism.In this study,PCR-mediated site-directed mutagenesis was used to find key amino acids in three regions Loop D,E and F of β2 subunit.The amino acid at one position of β2 subunit was replaced with the amino acid corresponding to the β4 subunit,and a total of 12 mutants were constructed.Simultaneously using the nAChR-specific agonist ACh and antagonists α-CTx(RegIIA and LvIA),the activity of mutants with ligand and the molecular mechanism of binding were studied by two-electrode voltage clamp technique.The results showed that when the 59th threonine(Thr,T)in Loop D of β2 subunit was mutated to lysine(Lys,K),although α3β3[T59K]mutant had no change in the activity for the agonist ACh,the activity of α3β3[T59K]for the antagonist α-CTx RegllA was significantly enhanced,and the half-inhibition concentration(IC50)decreased from 35 nM(wild-type α3β2 nAChR)to 3.1 nM,reduced by 11 times.α3β3[T59K]activity for the antagonist α-CTx LvIA was also significantly enhanced,and the half-inhibition concentration(IC50)decreased from 16 nM(wild-type α3β2 nAChR)to 3.2 nM,reduced by a factor of 5.In Loop E of β2 subunit,when the 106th phenylalanine(Phe,F)was mutated to valine(Val,V),the 108th serine(Ser,S)was mutated to threonine(Thr,T),the 111th valine(Val,V)was mutated to isoleucine(Ile,I)and the 113th serine(Ser,S)was mutated to arginine(Arg,R),the activity of α3β2[FIO6V]mutant for agonist ACh was increased after mutation,and the half effective concentration(EC50)was changed from 43μM to 22μM,increased by 2 times.The activities of α3β2[F106V],α3β2[S108T]andα3β2[V11I]nAChR for the antagonist α-CTx RegIIA were decreased,and the IC50s were increased from 35 nM to 360 nM,440 nM and 870 nM,reduced by 10 times,13 times,25 times respectively.But the activity of α3β2[S113R]was enhanced,and the IC50 was reduced from 35 nM(wild-type α3β2 nAChR)to 3.2 nM,enhanced by 11 times.The activities of α3β2[F106V],α3β2[S108T]and α3β2[V111I]nAChR for the antagonistα-CTx LvlA were decreased,and the IC50s was increased from 16 nM to 400 nM,1400 nM and 340 nM,respectively,and the activity was reduced by 25 times,88 times,21 times.On the contrary,the activity of α3β2[S113R]was enhanced,and the IC50 was reduced from 16 nM to 4.1 nM,enhanced by 4 times.When the serine(Ser,S)at position 168 in Loop F ofβ2 subunit was mutated to isoleucine(Ile,I),the activity of α3β2[S1681]mutant for the agonist ACh was enhanced,and the EC50 was changed from 43 μM to 26μM and the activity was increased by 1.6 times.The activity of α3β2[S1681]nAChR for the antagonistα-CTx ReglIA was increased,and the IC50 decreased from 35 nM to 1.0 nM,increased by 35 folds.The activity of α3β2[S1681]nAChR for the antagonist α-CTx LvlA was also increased,and the IC50 was decreased from 16 nM to 0.7 nM,increased by 23 times.In this study,conditions of PCR-mediated site-directed mutagenesis was optimized,12 mutants of α3β2 nAChR were established,and four new key amino acid sites 106,108,113,and 168 of β2 subunit were found.This study provides a new method for finding key amino acids on nAChR.In addition,the established mutant models provide a basis for studying the molecular mechanism of ligand interaction with α3β2 nAChR.It also provides a basis for studying the pharmacological mechanisms based on α3β2 nAChR. |
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