A role for Leu247 residue within transmembrane domain 2 in ginsenoside-mediated alpha7 nicotinic acetylcholine receptor regulation.
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.
Lee
Byung-Hwan
BH
Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University, Seoul 143-701, Korea.
Choi
Sun-Hye
SH
Pyo
Mi Kyung
MK
Shin
Tae-Joon
TJ
Hwang
Sung-Hee
SH
Kim
Bo-Ra
BR
Lee
Sang-Mok
SM
Lee
Jun-Ho
JH
Lee
Joon-Hee
JH
Lee
Hui Sun
HS
Choe
Han
H
Han
Kyou-Hoon
KH
Kim
Hyoung-Chun
HC
Rhim
Hyewhon
H
Yong
Joon-Hwan
JH
Nah
Seung-Yeol
SY
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
D036145:Ginsenosides; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; C097367:ginsenoside Rg3; D007930:Leucine