Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.

B.H White,J.B Cohen

doi:10.1016/s0021-9258(19)49602-6

Abstract

To characterize structural changes induced in the nicotinic acetylcholine receptor (AChR) by agonists, we have mapped the sites of photoincorporation of the cholinergic noncompetitive antagonist 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (]125I]TID) in the presence and absence of 50 microM carbamylcholine. [125I]TID binds to the AChR with similar affinity under both these conditions, but agonist inhibits photoincorporation into all subunits by greater than 75% (White, B. H., Howard, S., Cohen, S. G., and Cohen, J. B. (1991) J. Biol. Chem. 266, 21595-21607). [125I]TID-labeled sites on the beta- and delta-subunits were identified by amino-terminal sequencing of both cyanogen bromide (CNBr) and tryptic fragments purified by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reversed-phase high-performance liquid chromatography. In the absence of agonist, [125I]TID specifically labels homologous aliphatic residues (beta L-257, delta L-265, beta V-261, and delta V-269) in the M2 region of both subunits. In the presence of agonist, labeling of these residues is reduced approximately 90%, and the distribution of labeled residues is broadened to include a homologous set of serine residues at the amino terminus of M2. In the beta-subunit residues beta S-250, beta S-254, beta L-257, and beta V-261 are all labeled in the presence of carbamylcholine. This pattern of labeling supports an alpha-helical model for M2 with the labeled face forming the ion channel lumen. The observed redistribution of label in the resting and desensitized states provides the first direct evidence for an agonist-dependent rearrangement of the M2 helices. The efficient labeling of the resting state channel in a region capable of structural change also suggests a plausible model for AChR gating in which the aliphatic residues labeled by [125I]TID form a permeability barrier to the passage of ions. We also report increased labeling of the M1 region of the delta-subunit in the presence of agonist.

Highlights

Cholinergic noncompetitive antagonist 3-(trifluorome- subunits of Torpedo californica are known (Noda et al, 1982, thyl)-3-(rn-['261]iodophenyl)diazirine(['2SI]TID)in the 1983a, 1983b; Claudio et al, 1983), and antagonistsof acetylcholine receptor (AChR) presence andabsence of 50 PM carbamylcholine. [1261]- function havehelped establish which subunits,and which
Chern. 266, 21595-21607)[1.2SI]TID-labeled y and a-6 subunit interfaces form the twoagonist binding sites on the 8- and &subunits were identifiedambiyno- sites of the AChR (PedersenandCohen, 1990).Residues terminal sequencing of both cyanogen bromide (CNBr) contributing to these sites in the a-subunits lie within an and tryptic fragments purifiedby Tricine sodium do- amino-terminal region preceding the firstof four hydrophobic decyl sulfate-polyacrylamide gel electrophoresis fol- segments (Ml-M4) common to all AChR subunits
We have evaluate release of incorporated [1251]TID.Theresultsare shown that ["sI]TID labels a site on the AChR shown in Fig. 1.The p, y, and &subunit trypsindigests each formed by all subunits which is pharmacologically distinct showeda prominent rise in agonist-sensitive lZ5Irelease at from thatof previously characterized Noncompetitive antagonists (NCAs) such as histrion- cycle 13, while the P- and &subunits each showed an addiicotoxin and phencyclidine, compounds that appear to bind tional release a t cycle 9

Summary

EXPERIMENTAL PROCEDURES'

2, 6, and 20 have been found to alter the conductanceof the AChR Scales after additioonf agonist (20-100 ms) when AChR would Preliminary Characterization of ['"ZJTZD Photoincorporanot yetbe expected to have undergonceonversion to this state tion into the6-, y-, and 6-Subunits-Preliminary experiments (DiPaola et al, 1990) Under this condition,when some or all to identify sitesof agonist-sensitive ['2sI]TID photoincorporof the labeling might be into AChR in the open state[,'HIQA ationintothe P-, y-,and&subunitsevaluated cyanogen incorporates into thheydrophobic region M1 of the a-subunit bromide (CNBr)andtrypsin for theirabilitytogenerate rather thanM2. We have evaluate release of incorporated [1251]TID.Theresultsare shown that ["sI]TID labels a site on the AChR shown in Fig. 1.The p-, y-, and &subunit trypsindigests each formed by all subunits which is pharmacologically distinct showeda prominent rise in agonist-sensitive lZ5Irelease at from thatof previously characterized NCAs such as histrion- cycle 13, while the P- and &subunits each showed an addiicotoxin and phencyclidine, compounds that appear to bind tional release a t cycle 9.

Cycle Number

Fraction Number

SO r M carb

Findings

DISCUSSION