Probing the Structure of the Nicotinic Acetylcholine Receptor Ion Channel with the Uncharged Photoactivable Compound [3H]Diazofluorene

Abstract

The nicotinic acetylcholine receptor (AChR) 1 isolated from the electric organ of the marine elasmobranch Torpedo californica is the best characterized member of a family of ligandgated ion channels which includes the ␥-aminobutyric acid, glycine, and serotonin 5-HT 3 receptors (for recent reviews, see Refs. 1-3). The AChR is composed of four homologous subunits (␣ 2 ␤␥␦) arranged quasi-symmetrically around a central cationselective ion channel (4). The subunits each have a characteristic topology as follows: a large hydrophilic N-terminal domain containing the agonist binding sites, followed in the primary structure by three hydrophobic membrane-spanning segments (M1-M3), a cytoplasmic domain, a fourth hydrophobic transmembrane segment (M4), and a short extracellular C-terminal tail.Noncompetitive antagonists (NCAs) are agents that block the AChR permeability response without binding to the agonist site. These compounds are structurally diverse and include many aromatic amines but also general anesthetics, steroids, and even the neuropeptide substance P (reviewed in Ref. 5). A number of NCAs have been instrumental in identifying regions of the AChR which contribute to the formation of the pore of the ion channel. Photoaffinity labeling studies with [ 3 H]chlorpromazine (6 -9) and [ 3 H]triphenylmethylphosphonium (10) as well as reaction with [ 3 H]meproadifen mustard (11) have all identified labeled residues within the M2 segments that would all lie on a common side of an ␣-helix. These results, in conjunction with the observed functional properties of AChRs with mutations in the M2 segments (1, 2), provide the basis for a model of the ion channel comprised of M2 segments of each subunit arranged as transmembrane ␣-helices around the central axis, a model consistent with studies of AChR three-dimensional structure derived from electron micrographic image analysis (4, 12). 125 I]TID labeled ␦Leu-265 and ␦Val-269 and the homologous residues in the other subunits that are located 9 and 13 amino acids to the C-terminal side of the conserved lysine residue at the N terminus of the M2 region (positions 9 and 13). In the desensitized state, the pattern of [ 125 I]TID-labeled residues broadened to include homologous serine residues at positions 2 and 6 (i.e. ␦Ser-258 and ␦Ser-

Structure and Function of the Glycine Receptor and Related Nicotinicoid Receptors

“…The M2 region alone is sufficient to form a chloride-conducting pore because synthetic M2-based peptides form Cl Ϫ -conducting channels when added to cells (24). Receptor topology was also probed using photoactivable lipophilic compounds to examine the protein-lipid interface of the nAChR (25). The periodicity of the labeled residues in M4 was consistent with a transmembrane ␣-helix with a face of the helix exposed to the acyl chains of the bilayer.…”

Tryptophan-scanning Mutagenesis in the αM3 Transmembrane Domain of the Muscle-type Acetylcholine Receptor

“…states, therefore suggesting that these domains display a substantial conformational change upon agonist-induced activation of the AChR (10,12,13,38,39). A recent study reported structural and conformational changes in the third transmembrane domain of a homologous receptor, the ␣M3 domain of the ␥-aminobutyric acid type A receptor (40).…”

Functional Reconstitution and Characterization of Recombinant Human α1-Glycine Receptors

“…Additionally, residues localized to the transmembrane domains of these receptors have been identified by cysteine scanning methods (for review see Ref. 22) as well as using lipophilic cross-linking agents (23)(24)(25). However, despite these and other studies on nAchR and other members of this neuroreceptor superfamily, the details of the molecular architecture of these ion channels remain refractory and subject to debate (for review see Ref.…”