A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated α7 nicotinic acetylcholine receptor regulation

Abstract

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of m~å~ñ ginseng, inhibit subsets of nAChR channel currents, but not α7, expressed in uÉåçéìë= ä~Éîáë oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of α7 nAChR induces alterations in channel gating properties and converts α7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(p)-ginsenoside Rg3 (Rg3) activity against the α7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg3. We further characterized Rg3 regulation of L247T receptors. We found that Rg3 inhibition of mutant α7 nAChR channel currents was reversible and concentration-dependent. Rg3 inhibition was strongly voltage-dependent and noncompetitive mannerK These results indicate that the interaction between Rg3 and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg3 interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg3 forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg3 localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for Rg3 at the channel pore