Abstract: α6/α3β2β3 nicotinic acetylcholine receptors (nAChRs) subtype is widely expressed in mesolimbic dopaminergic pathway and other regions, which plays a vital role in modulating the release of multiple neurotransmitters and drug reward mechanisms. It has become a potential therapeutic target for the treatment of various neuropsychiatric disorders and drug addiction. However, most ligands identified with high affinity and selectivity targeting α6/α3β2β3 subtype showed limited subtype discrimination against the closely related α3β2 subtype. Furthermore, the molecular mechanisms underlying ligands interact with α6/α3β2β3 nAChR remain absent. In this study, the related approaches of site-directed mutagenesis of receptor, in vitro transcription and electrophysiological assay were utilized to establish the drug screening models of wild-type α6/α3β2β3 nAChR and its α6/α3 subunit single-point mutants. The effects led by single amino acid substitution to the function of α6/α3β2β3 nAChR were also evaluated. The animal experimental protocol involved in this study has been approved by the Ethics Committee of Guangxi University (Approval No: GXU-2023-0249). As the results showed, the half maximal effective concentration (EC₅₀) for ACh activating α6/α3β2β3 nAChR was 18.5 μmol·L^-1. All 7 single-point mutations of α6/α3 subunit resulted in increase of the agonist sensitivity, while the EC₅₀ of ACh towards mutants α6/α3V112Eβ2β3、α6/α3T147Sβ2β3、α6/α3E152Kβ2β3 nAChR decreased to the greatest extent (EC₅₀s were 3.5, 4.4, and 2.0 μmol·L^-1, respectively, which were 0.1-0.2-fold relative to that of the wild-type receptor). These results established models for the research of structure-function relationship of α6/α3β2β3 nAChRs and molecular mechanism of ligand-receptor specific binding, as well as provided theoretical foundation for the design and optimization of selective drug leads targeting α6/α3β2β3 nAChRs.