Molecular Basis For Differential Activities Of Five α4/7-Conotoxins At α3β2 And α4β2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors（nAChRs）are members of the"Cys-loop"family of pentameric ligand-gated ion channels（LGIC）.The endogenous ligand acetylcholine binds to the N-terminal extracellular domain of nAChRs,which regulates the opening and closing of receptor channels.There are many subtypes of nAChRs,among whichα3β2 andα4β2 nAChRs are important neuronal nAChRs,which play important physiological functions in the organism.α4β2 nAChR is a valuable pharmacological target that correlates with nicotine addiction,age-associated memory impairment and cognitive disorders.α3β2nAChR,mainly distributing in dorsal root ganglion and spinal cord,is involved in the regulation of pain sensation.α-Conotoxins（α-CTxs）are natural biotoxins that specifically target nAChRs and can be used as molecular probes to study the physiological and pathological functions of different subtypes of nAChRs,or directly developed as medicines.It has been found that a variety ofα-CTxs can strongly blockα3β2 nAChR,but their activities onα4β2 nAChR are very weak or almost inactive.Sinceα3β2 andα4β2 nAChRs both containβ2 subunit,andα3 andα4 subunits have high homologous similarity,it is speculated that some key sites inα3 andα4 subunits play key roles in activity difference.The purpose of this study was to explore the interaction between variousα4/7-CTxs and agonists and mutantα3β2 andα4β2nAChRs by receptor mutation technology,and to analyze the molecular mechanism of their interaction.At the same time,it would have important guiding significance for the design ofα4β2 nAChR specific antagonists.Based on the homologous similarity betweenα3 andα4 subunits,this study applied PCR-mediated site-directed mutagenesis to perform on several amino acid sites in the N-terminal extracellular domain ofα3 andα4 subunits,and 9 single-point mutant receptors and 2 double-point mutant receptors were successfully constructed.By using two-electrode voltage clamp,5 naturalα4/7-CTxs that can efficiently and specifically blockα3β2receptor were selected as ligands to study the interaction between these conotoxins and mutant subunits,and elucidate the molecular mechanism.The results of electrophysiology experiment showed that compared to wild-typeα3β2 receptor,the activities of GIC,GID,Lt IA,Lv IA and Reg IIA toα3（I188R）β2 andα3（Q198P）β2 receptors decreased visibly,when Ile-188 and Gln-198 in theα3 subunit were mutated to arginine and proline.The activity of GID toα3（E194A）β2 receptor decreased by 13.02-fold,when Glu-194 in theα3subunit was mutated to alanine.The activity of Lt IA toα3（N191E）β2 receptor decreased by 16.71-fold,when Asn-194 in theα3 subunit was mutated to glutamate.The activities of GIC,GID and Lv IA toα3（I188R,Q198P）β2 receptor respectively decreased by 58.27-fold,64.71-fold and 135.96-fold,and the IC₅₀ values of Lt IA and Reg IIA onα3（I188R,Q198P）β2 receptor both were over 10,000 n M.For wild-typeα4β2 receptor,the IC₅₀values of GIC,Lt IA,Lv IA and Reg IIA were far over 10,000 n M and the IC₅₀ value of GID was 950.2 n M.When Arg-188 and Pro-198 in theα4 subunit were mutated to isoleucine and glutamine,the IC₅₀ values of GIC,GID and Lv IA onα4（R188I）β2 receptor were reduced to 132.2,260.2 and 7205 n M,respectively,the IC₅₀ values of GIC,GID,Lv IA and Reg IIA onα4（P198Q）β2 receptor were reduced to 133.7,135.2,2619 and 1349 n M,respectively.The activities of 5 naturalα4/7-CTxs onα4（R188I,P198Q）β2 receptors were greatly improved compared to wild-typeα4β2 receptor.The IC₅₀ values of GIC,GID,Lt IA,Lv IA and Reg IIA onα4（R188I,P198Q）β2 receptors were reduced to 8.550,62.64,1461,210.2 and 131.6 n M,respectively.According to the results of above study,we also tested the effects of agonists such as acetylcholine,nicotine,cytisine and varenicline on the function of mutant nAChRs.The results of electrophysiology experiment showed that the acetylcholine concentration of50%maximal effect(EC₅₀)for wild-typeα4β2,α4（R188I）β2,α4（P198Q）β2 andα4（R188I,P198Q）β2 nAChRs were 67.15,63.83,22.43 and 1.368μM,respectively.The EC₅₀ values of nicotine for wild-typeα4β2,α4（R188I）β2,α4（P198Q）β2 andα4（R188I,P198Q）β2nAChRs was 1.431,0.6274,1.046 and 0.2678μM,respectively.Compared with the peak current amplitude induced by ACh（10 m M）,the maximum efficacy（Emax）of cytisine for wild-typeα4β2 andα4（R188I,P198Q）β2 nAChRs was increased from 37.62%to 95.14%.The Emax of varenicline for wild-typeα4β2 andα4（R188I,P198Q）β2 nAChRs was increased from 67.94%to 94.69%.As the results of this study showed,it was closely related to Arg-188 and Pro-198 ofα4 subunit thatα4/7-CTxs had strong activity onα3β2 nAChR but weak activity onα4β2nAChR.In addition,we also found Glu-194 was an important site for the binding of GID toα3 subunit,and Asn-191 was an important site for the binding of Lt IA toα3 subunit.The results of study on the interaction between agonists and mutant receptors showed that the mutation of Arg-188 and Pro-198 ofα4 subunit to isoleucine and glutamine not only enhanced the sensitivities ofα4β2 nAChR to ACh and nicotin,but also significantly improved the channel opening efficiencies ofα4β2 nAChR to cytisine and varenicline.The above studies clearly clarified molecular basis of the difference in the activities of 5α4/7-CTxs onα3β2 andα4β2 nAChRs,which provided foundation for the design ofα-CTxs mutants acting onα4β2 nAChR and can be used to guide the design and structural optimization of nAChRs agonists.