Identifying the lipid-protein interface of the alpha4beta2 neuronal nicotinic acetylcholine receptor: hydrophobic photolabeling studies with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine.
Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID photoincorporated into both alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [125I]TID labeled homologous amino acids alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 segment, [125I]TID labeled residues Cys226 and Cys231, which correspond to the [125I]TID-labeled residues Cys222 and Phe227 at the lipid-exposed face of the Torpedo alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.
Hamouda
Ayman K
AK
Department of Pharmacology and Neuroscience, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Sanghvi
Mitesh
M
Chiara
David C
DC
Cohen
Jonathan B
JB
Blanton
Michael P
MP
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D015335:Molecular Probes; D011978:Receptors, Nicotinic; C427403:alpha(4)beta(4) nicotinic receptor