title: A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated alpha7 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 Panax ginseng, inhibit subsets of nAChR channel currents, but not alpha7, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of alpha7 nAChR induces alterations in channel gating properties and converts alpha7 nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside Rg(3) (Rg(3)) activity against the alpha7 nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to Rg(3). We further characterized Rg(3) regulation of L247T receptors. We found that Rg(3) inhibition of mutant alpha7 nAChR channel currents was reversible and concentration-dependent. Rg(3) inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between Rg(3) and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in Rg(3) interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that Rg(3) forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas Rg(3) 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 Rg(3) at the channel pore.
authors.affiliation: Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea.
authors.forename: Byung-Hwan
authors.initials: BH
authors.lastname: Lee
authors.forename: Sun-Hye
authors.initials: SH
authors.lastname: Choi
authors.forename: Mi Kyung
authors.initials: MK
authors.lastname: Pyo
authors.forename: Tae-Joon
authors.initials: TJ
authors.lastname: Shin
authors.forename: Sung-Hee
authors.initials: SH
authors.lastname: Hwang
authors.forename: Bo-Ra
authors.initials: BR
authors.lastname: Kim
authors.forename: Sang-Mok
authors.initials: SM
authors.lastname: Lee
authors.forename: Jun-Ho
authors.initials: JH
authors.lastname: Lee
authors.forename: Joon-Hee
authors.initials: JH
authors.lastname: Lee
authors.forename: Hui Sun
authors.initials: HS
authors.lastname: Lee
authors.forename: Han
authors.initials: H
authors.lastname: Choe
authors.forename: Kyou-Hoon
authors.initials: KH
authors.lastname: Han
authors.forename: Hyoung-Chun
authors.initials: HC
authors.lastname: Kim
authors.forename: Hyewhon
authors.initials: H
authors.lastname: Rhim
authors.forename: Joon-Hwan
authors.initials: JH
authors.lastname: Yong
authors.forename: Seung-Yeol
authors.initials: SY
authors.lastname: Nah
mesh_terms: D000494:Allosteric Regulation / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens / Q000235:genetics; D003198:Computer Simulation; D036145:Ginsenosides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D005894:Panax; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011978:Receptors, Nicotinic / Q000737:chemistry; D015398:Signal Transduction / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D014982:Xenopus laevis / Q000235:genetics; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D036145:Ginsenosides; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C097367:ginsenoside Rg3; D007930:Leucine