Probing the structure of the affinity-purified and lipid-reconstituted torpedo nicotinic acetylcholine receptor.
The Torpedo nicotinic acetylcholine receptor (nAChR) is the only member of the Cys-loop superfamily of ligand-gated ion channels (LGICs) that is available in high abundance in a native membrane preparation. To study the structure of the other LGICs using biochemical and biophysical techniques, detergent solubilization, purification, and lipid reconstitution are usually required. To assess the effects of purification on receptor structure, we used the hydrophobic photoreactive probe 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) to compare the state-dependent photolabeling of the Torpedo nAChR before and after purification and reincorporation into lipid. For the purified nAChR, the agonist-sensitive photolabeling within the M2 ion channel domain of positions M2-6, M2-9, and M2-13, the agonist-enhanced labeling of deltaThr274 (deltaM2-18) within the delta subunit helix bundle, and the labeling at the lipid-protein interface (alphaMu4) were the same as for the nAChR in native membranes. However, addition of agonist did not enhance [(125)I]TID photolabeling of deltaIle288 within the deltaM2-M3 loop. These results indicate that after purification and reconstitution of the Torpedo nAChR, the difference in structure between the resting and desensitized states within the M2 ion channel domain was preserved, but not the agonist-dependent change of structure of the deltaM2-M3 loop. To further characterize the pharmacology of [(125)I]TID binding sites in the nAChR in the desensitized state, we examined the effect of phencyclidine (PCP) on [(125)I]TID photolabeling. PCP inhibited [(125)I]TID labeling of amino acids at the cytoplasmic end of the ion channel (M2-2 and M2-6) while potentiating labeling at M2-9 and M2-13 and allosterically modulating the labeling of amino acids within the delta subunit helix bundle.
Hamouda
Ayman K
AK
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Chiara
David C
DC
Blanton
Michael P
MP
Cohen
Jonathan B
JB
D000345:Affinity Labels; D000818:Animals; D001389:Azirines / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D020355:Cholates / Q000737:chemistry; D007457:Iodine Radioisotopes / Q000737:chemistry; D007473:Ion Channels / Q000378:metabolism; D050356:Lipid Metabolism; D008958:Models, Molecular; D018733:Nicotinic Antagonists / Q000378:metabolism / Q000494:pharmacology; D010622:Phencyclidine / Q000378:metabolism / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared; D013194:Staining and Labeling; D013379:Substrate Specificity; D014101:Torpedo
D000345:Affinity Labels; D001389:Azirines; D020355:Cholates; D007457:Iodine Radioisotopes; D007473:Ion Channels; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D010622:Phencyclidine