| Theα6/α3β4 subtype is a special nicotinic acetylcholine receptors（nAChRs）.Theα6*nAChRs is difficult to express in vitro.As a result of this,there are few researches about the interaction between ligands andα6β4*subtypes at the molecular level.Recent studies have found that they expressed in rat and mouse dorsal root ganglia（DRG）,and they are distributed in the catecholamine nerve pathway,which is closely related to the pathogenesis of many diseases.The study ofα6*nAChRs is critical for further revealing the structure and pharmacological activities of the receptors.α-conotoxin TxIB is an antagonist towards ratα6/α3β2β3 nAChRs and humanα6/α3β4 nAChR,whose IC50 towards humanα6/α3β4 nAChR is 30 times of the ratsα6/α3β4 nAChR.[D1G,ΔQ14]LvIC is highly specific to human or ratα6β4*nAChR,and has no effects on other receptors.The amino acid sequences of human and ratα6/α3β4 nAChRs are different.The human and rat mutantα6/α3β4 nAChRs were constructed by site-directed mutagenesis of the extracellular N-terminal domain,which were also regarded as probes to identify the biological activity ofα6/α3β4 nAChRs.Meanwhile,the key sites of ligand interaction withα6/α3β4 nAChRs were explored and the molecular mechanism of the interaction between the receptor and the ligand was clarified.It was found that the sensitivity of ratα6/α3β4[S52N,I53V]to ACh was increased,and the EC50was 59.58μmol·L-1,which is 1/2 of the wild type.The inhibitory activity ofα-conotoxin TxIB on humanα6/α3[M112V]β4 was weaker than that of wild-typeα6/α3β4,and the inhibitory activity of TxIB on ratα6/α3β4[K56D]is significantly enhanced,which is equivalent to that of human wild-typeα6/α3β4 nAChR.Therefore,it can be inferred that the 112th amino acid of theα6 subunit and the 56th amino acid of theβ4 subunit are likely to be the key sites for TxIB to act on theα6/α3β4 nAChR,which is of great significance for the subsequent structural modification of TxIB and clarification of the structure and function ofα6/α3β4 nAChRs. |
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