Structural studies with affinity-purified muscle and neuronal nicotinic acetylcholine receptors
Nicotinic acetylcholine receptors (nAChRs) are a heterogeneous group of ligand gated ion channels that mediate the function of the endogenous neurotransmitter acetylcholine. A more refined understanding of the molecular structure of individual neuronal nAChRs is a prerequisite for the development of subtype-selective ligands. Such ligands will be valuable in the understanding and treatment of certain CNS diseases as well as nicotine addiction. Using an acetylcholine derivatized affinity column originally used for purification of Torpedo nAChR, we have purified a4b2 and a4b4 neuronal nAChRs from HEK293 cell lines that stably express a4b2 or a4b4 nAChRs respectively. The lipid-protein interface and the agonist-binding site of purified neuronal nAChRs were directly examined using photoaffinity labeling with [125I]TID and [125I]Epibatidine respectively. The preliminary results of these studies include: 1) [125I]TID photoincorporates into the M4 and M1 transmembrane segments of both a4 and b2 subunits; 2) Within a4M4/b2M4 segments, [125I]TID labeled amino acid residues a4Cys582/b2Cys445 which are identical (in the aligned sequence) to the [125I]TID-labeled residues Cys418 in the lipid exposed face of Torpedo a1M4; 3) [125I]TID also labeled a4Arg572 within a4M4 which is situated in the same position as His408 in the lipid exposed face of Torpedo a1M4; 4) Within a4M1 segment, [125I]TID labeled Cys226, Cys231 and Leu232 which correspond to [125I]TID labeled residues Cys222, Phe227 and Leu228 in the lipid exposed face of Torpedo a1M1. In similar fashion, [125I]TID labeled b2Cys220 and b2Val221 which correspond to [125I]TID labeled Cys222 and Leu223 in the lipid exposed face of Torpedo a1M1. 5) Further studies are needed to determine if [125I]TID photoincorporates into the M2 and M3 transmembrane segments; 6) Photoaffinity labeling of a4b2 and a4b4 nAChRs with [125I]Epibatidine reveals that [125I]Epibatidine photoincorporates specifically into the b4 subunit with no (or insignificant photoincorporation) within a4 and b2 subunits; 7) [125I]Epibatidine photoincorporation ion the a4 subunit was limited to the agonist binding pocket as the majority of labeling was displaceable by addition of nicotine; 8) The site of specific [125I]Epibatidine photincorporation within the b4 subunit was mapped to loop E (b4Val102-Glu131) within the complementary components of the agonist binding site.