Mapping the lipid-exposed regions in the Torpedo californica nicotinic acetylcholine receptor

Abstract

To identify regions of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used 1-azidopyrene (1-AP) as a fluorescent, photoactivatable hydrophobic probe. For AchR-rich membranes equilibrated with 1-AP, irradiation at 365 nm resulted in covalent incorporation in all four AchR subunits with each of the subunits incorporating approximately equal amounts of label. To identify the regions of the AchR subunits that incorporated 1-AP, subunits were digested with Staphylococcus aureus V8 protease and trypsin, and the resulting fragments were separated by SDS-PAGE followed by reverse-phase high-performance liquid chromatography. N-terminal sequence analysis identified the hydrophobic segments M1, M3, and M4 within each subunit as containing the sites of labeling. The labeling pattern of 1-AP in the alpha-subunit was compared with that of another hydrophobic photoactivatable probe, 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID). The nonspecific component of [125I]TID labeling [White, B., Howard, S., Cohen, S. G., & Cohen, J.B. (1991) J. Biol. Chem. 266, 21595-21607] was restricted to the same regions as those labeled by 1-AP. The [125I]TID residues labeled in the hydrophobic segment M4 were identified as Cys-412, Met-415, Cys-418, Thr-422, and Val-425. The periodicity and distribution of labeled residues establish that the M4 region is alpha-helical in nature and indicate that M4 presents a broad face to membrane lipid.

Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: Electrophysiology-coordinated photochemistry and mass spectrometry

“…Table 1 lists mass accuracies of the detected photolabeled peptides. Some photolabeled peptides deviated markedly from (46,47,55). C427 and T431 (shown in bold underlined type with red͞green circles) were identified by TID incorporation both in previous studies and in the present study.…”

“…4 illustrate that M4 photolabeled residues identified by this study reside on the same helical face as residues identified in previously published reports. Thus the present results further define the lipid-exposed face of the M4 transmembrane ␣-helix (46,47).…”

Coupled Proteolytic and Mass Spectrometry Studies Indicate a Novel Topology for the Glycine Receptor

“…No internal cleavages were detected within this region (although this region is primarily hydrophobic and offers few potential targets for proteolysis). Given the reported pattern of lipophilic photoactivable reagent incorporation within the M4 region of nAchR (32), the presence of a phosphorylation site at Ser-391, and the extracellular location of the C terminus, it is expected that a single TM ␣-helix is located within residues 392-416.…”

Tryptophan Substitutions at Lipid-exposed Positions of the Gamma M3 Transmembrane Domain Increase the Macroscopic Ionic Current Response of the Torpedo californica Nicotinic Acetylcholine Receptor

“…The M2 and M1 segments from each subunit associate around a central axis to form part of the aqueous ion channel. In contrast, the M4 and M3 segments have been shown to have the largest contact with lipid (Blanton & Cohen, 1992.…”