Identifying the binding site of novel methyllycaconitine (MLA) analogs at α4β2 nicotinic acetylcholine receptors.
Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4β2, and α3β4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 μM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4β2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4β2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)(3)(β2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.
Faculty of Pharmacy, The University of Sydney, Australia.
Gracia X J
GX
Quek
Diana
D
Lin
Jill I
JI
Halliday
Nathan
N
Absalom
Joseph I
JI
Ambrus
Andrew J
AJ
Thompson
Martin
M
Lochner
Sarah C R
SC
Lummis
Malcolm D
MD
McLeod
Mary
M
Chebib
D000157:Aconitine / Q000031:analogs & derivatives* / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D008564:Membrane Potentials; D008956:Models, Chemical; D008958:Models, Molecular; D015394:Molecular Structure; D009150:Mustard Compounds / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor
C579165:Chrna7 protein, rat; D009150:Mustard Compounds; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; C093171:methanethiosulfonate ethylammonium; C403530:nicotinic receptor alpha3beta4; C403531:nicotinic receptor alpha4beta2; C054634:methyllycaconitine; D005020:Ethyl Methanesulfonate; D003545:Cysteine; D000157:Aconitine