Construction Of α3~* Nicotinic Acethlcholine Receptor Mutants And Study Of Their Function

Objective:By exchange the extracellular N-terminal domain’s non-homologous amino acids of a3 subunit to corresponding amino acids of a6 subunit,and construct the point mutants of α3*nicotine acetylcholine receptors.Finding the key amino acids is important to research the interaction molecular mechanism between α3*nAChRs and ligands.Methods:Primer premier 5.0 was used to design the mutants primers.The α3 nAChRs subunit was used as template and mutants were constructed by PCR mediated site-directed mutation techniques.Point mutated primers were designed according to rat α3 subunit gene.The RNA of α3 subunit point mutants were synthesized by in vitro transcription.The expression of mutants in Xenopus oocytes were detected by two-electrode voltage-clamp techniques.Gating properties and receptor’s activity of the mutants were detected by agonist ACh and antagonist α-CTx RegIIA.Results:Twenty kinds of mutants of α3β2 subtypes were constructed successfully.The EC50 of agonist ACh to wild-type α3(β2 nAChRs is 54.82 μM,to α3(K152E)β2 nAChRs is 5.711 μM,the EC50 of wild-type α3β2 is 9.5 times higher than α3(K152E)β2 nAChRs,means the increased activity was observed in α3(K152E)β2 nAChRs.The mutants α3(Y167F)β2 nAChRs andα3(S170N)β2 nAChRs can’t be evoked by any concentration of ACh,means those two mutants lose the sensibility to agonist Ach.The EC50 of agonist ACh to wild-type α3β2 nAChRs and other mutants were within the five times,means the agonist ACh to those mutants’ activity were similar to wild-type α3β2 nAChRs.It is indicated that the 152th,167th and 170 th amino acid of the α3 subunit are the key binding sites of agonist ACh.However,the IC50 of antagonist α-CTx RegIIA to wild-type α3β2 is 33.77 nM,toα3(K152E)β2 is 233,1 nM,the IC50 of α3(K152E)β2 is 6.9 times higher than wild-typeα3β2 nAChRs,means the decreased activity was observed in α3(K152E)β2 nAChRs.The IC50 of antagonist α-CTx RegIIA to α3(E184D)β2 is 0.8 nM,the IC50 of wild-type α3β2 is 42 times higher than α3(E184D)β2 nAChRs,means the increased activity was observed inα3(E184D)β2 nAChRs.The IC50 of antagonist α-CTx RegIIA to α3(Q195T)β2 is 10.50 μM,the IC50 of α3(Q195T)β2 greater than 10 μM,means the loss-of-function was observed inα3(Q195T)β2 nAChRs.The IC50 of antagonist α-CTx RegIIA to wild-type α3β2 nAChRs and other mutants were within five times,means those mutants’ activity of RegIIA were similar to wild-type α3β2 nAChRs.It is indicated that the 152th,184th and 195th amino acid of the α3 subunit are the key binding sites of α-CTx RegIIA.Conclusion:After change the amino acid on a3 subunit extracellular N-terminal domain to corresponding amino acids of α6 subunit,the α3 subunit mutations were constructed successfully.In this study we have found the key amino acid on α3 subunit which can influence the binding activity of agonist ACh and antagonist α-CTx RegIIA.This can provide a function model to make more other receptor mutants,and would be helpful to interrogate the interaction between drug and α3*nAChRs.