Construction Of ?3~* 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 ?3*nicotine acetylcholine receptors.Finding the key amino acids is important to research the interaction molecular mechanism between ?3*nAChRs and ligands.Methods:Primer premier 5.0 was used to design the mutants primers.The ?3 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 ?3 subunit gene.The RNA of ?3 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 ?-CTx RegIIA.Results:Twenty kinds of mutants of ?3?2 subtypes were constructed successfully.The EC50 of agonist ACh to wild-type ?3(?2 nAChRs is 54.82 ?M,to ?3(K152E)?2 nAChRs is 5.711 ?M,the EC50 of wild-type ?3?2 is 9.5 times higher than ?3(K152E)?2 nAChRs,means the increased activity was observed in ?3(K152E)?2 nAChRs.The mutants ?3(Y167F)?2 nAChRs and?3(S170N)?2 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 ?3?2 nAChRs and other mutants were within the five times,means the agonist ACh to those mutants' activity were similar to wild-type ?3?2 nAChRs.It is indicated that the 152th,167th and 170 th amino acid of the ?3 subunit are the key binding sites of agonist ACh.However,the IC50 of antagonist ?-CTx RegIIA to wild-type ?3?2 is 33.77 nM,to?3(K152E)?2 is 233,1 nM,the IC50 of ?3(K152E)?2 is 6.9 times higher than wild-type?3?2 nAChRs,means the decreased activity was observed in ?3(K152E)?2 nAChRs.The IC50 of antagonist ?-CTx RegIIA to ?3(E184D)?2 is 0.8 nM,the IC50 of wild-type ?3?2 is 42 times higher than ?3(E184D)?2 nAChRs,means the increased activity was observed in?3(E184D)?2 nAChRs.The IC50 of antagonist ?-CTx RegIIA to ?3(Q195T)?2 is 10.50 ?M,the IC50 of ?3(Q195T)?2 greater than 10 ?M,means the loss-of-function was observed in?3(Q195T)?2 nAChRs.The IC50 of antagonist ?-CTx RegIIA to wild-type ?3?2 nAChRs and other mutants were within five times,means those mutants' activity of RegIIA were similar to wild-type ?3?2 nAChRs.It is indicated that the 152th,184th and 195th amino acid of the ?3 subunit are the key binding sites of ?-CTx RegIIA.Conclusion:After change the amino acid on a3 subunit extracellular N-terminal domain to corresponding amino acids of ?6 subunit,the ?3 subunit mutations were constructed successfully.In this study we have found the key amino acid on ?3 subunit which can influence the binding activity of agonist ACh and antagonist ?-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 ?3*nAChRs.