To identify amino acid residues of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used the photoactivatable, hydrophobic probe 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine([125I]TID). The pattern of [125I]TID incorporation into the M3 and M4 hydrophobic segments of each subunit was the same both in the presence and absence of the agonist carbamoylcholine and in the presence of an excess of nonradioactive TID, consistent with nonspecific photoincorporation from the lipid-protein interface. [125I]TID reacted with five residues in alpha-M4 [Blanton, M.P., & Cohen, J. B. (1992) Biochemistry 31, 3738-3750] but with only two or three residues in M4 segments of beta-, gamma-, and delta-subunits. In delta-M3, [125I]TID reacted with Met-293, Ser-297, Gly-301, Val-304, and Asn-305 as well as with Ile-288 preceding M3. Residues at corresponding positions were labeled in beta-M3 (Met-285, Ile-289, Phe-293) and in gamma-M3 (Phe-292, Leu-296, Met-299, and Asn-300) as well as gamma-Ile-283. Within alpha-M3, Phe-284 and Ser-287 were labeled. The periodicity of labeled residues provides the first direct evidence that M3 as well as M4 segments of each subunit are organized as transmembrane alpha-helices each with substantial contact with lipid. In addition, in alpha-M1 [125I]TID reacted nonspecifically with Cys-222, Leu-223, Phe-227, and Leu-228, a pattern of incorporation inconsistent with the labeling pattern expected either for a "face" of an alpha-helix or a beta-sheet.

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.

The structural and functional properties of reconstituted nicotinic acetylcholine receptor membranes composed of phosphatidyl choline either with or without cholesterol and/or phosphatidic acid have been examined to test the hypothesis that receptor conformational equilibria are modulated by the physical properties of the surrounding lipid environment. Spectroscopic and chemical labeling data indicate that the receptor in phosphatidylcholine alone is stabilized in a desensitizedlike state, whereas the presence of either cholesterol or phosphatidic acid favors a resting-like conformation. Membranes that effectively stabilize a resting-like state exhibit a relatively large proportion of non-hydrogen-bonded lipid ester carbonyls, suggesting a relatively tight packing of the lipid head groups and thus a well ordered membrane. Functional reconstituted membranes also exhibit gel-to-liquid crystal phase transition temperatures that are higher than those of nonfunctional reconstituted membranes composed of phosphatidylcholine alone. Significantly, incorporation of the receptor into phosphatidic acid-containing membranes leads to a dramatic increase in both the lateral packing densities and the gel-to-liquid crystal phase transition temperatures of the reconstituted lipid bilayers. These results suggest a functional link between the nicotinic acetylcholine receptor and the physical properties of phosphatidic acid-containing membranes that could underlie the mechanism by which this lipid preferentially enhances receptor function.

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[ 125 I]iodophenyl) diazirine ([ 125 I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA) and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e. functionality). The present studies demonstrate that: (1) there is no obligatory requirement for PC; (2) that increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, this effect saturates at ∼ 35 mol% (molar lipid percentage); (3) the effect of increasing levels of PA saturates at ∼12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e. nonfunctional). Native Torpedo membranes contain ∼35 mol% CH but less than 1 mol% PA, suggesting that other anionic lipid may substitute for PA. We report that: (4) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (∼50-60%) compared to PA; (5) For nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below ∼65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid-and sterol-(CH) protein interactions.The agonist-induced conformational transitions that underlie the function of the Torpedo nicotinic acetylcholine receptor (nAChR) have been shown to be highly dependent on the surrounding lipid environment, providing a model system for understanding lipid-protein interactions (1-3). The nAChR is the best characterized member of a superfamily of ligandgated ion channels (LGIC) that includes muscle-and neuronal-type nAChRs, serotonin 5-HT 3 receptors, gamma-amino butyric acid type A receptors (GABA A ), and glycine receptors (4,5). The Torpedo (muscle-type) nAChR is a pentameric multispanning transmembrane protein comprised of four homologous subunits with a subunit stoichiometry of 2α:β:γ:δ that are arranged pseudosymetrically around a central axis forming an ion-conducting channel. In

The photoactivatable sterol probe [3alpha-(3)H]6-Azi-5alpha-cholestan-3beta-ol ([3H]Azicholesterol) was used to identify domains in the Torpedo californica nicotinic acetylcholine receptor (nAChR) that interact with cholesterol. [3H]Azicholesterol partitioned into nAChR-enriched membranes very efficiently (>98%), photoincorporated into nAChR subunits on an equal molar basis, and neither the pattern nor the extent of labeling was affected by the presence of the agonist carbamylcholine, consistent with photoincorporation at the nAChR lipid-protein interface. Sites of [3H]Azicholesterol incorporation in each nAChR subunit were initially mapped by Staphylococcus aureus V8 protease digestion to two relatively large homologous fragments that contain either the transmembrane segments M1-M2-M3 (e.g., alphaV8-20) or M4 (e.g., alphaV8-10). The distribution of [3H]Azicholesterol labeling between these two fragments (e.g., alphaV8-20, 29%; alphaV8-10, 71%), suggests that the M4 segment has the greatest interaction with membrane cholesterol. Photolabeled amino acid residues in each M4 segment were identified by Edman degradation of isolated tryptic fragments and generally correspond to acidic residues located at either end of each transmembrane helix (e.g., alphaAsp-407). [3H]Azicholesterol labeling was also mapped to peptides that contain either the M3 or M1 segment of each nAChR subunit. These results establish that cholesterol likely interacts with the M4, M3, and M1 segments of each subunit, and therefore, the cholesterol binding domain fully overlaps the lipid-protein interface of the nAChR.

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-

The topography of nicotinic acetylcholine receptor (AChR) membrane-embedded domains and the relative affinity of lipids for these protein regions were studied using fluorescence methods. Intact Torpedo californica AChR protein and transmembrane peptides were derivatized with N-(1-pyrenyl)maleimide (PM), purified, and reconstituted into asolectin liposomes. Fluorescence mapped to proteolytic fragments consistent with PM labeling of cysteine residues in ␣M1, ␣M4, ␥M1, and ␥M4. The topography of the pyrene-labeled Cys residues with respect to the membrane and the apparent affinity for representative lipids were determined by differential fluorescence quenching with spin-labeled derivatives of fatty acids, phosphatidylcholine, and the steroids cholestane and androstane. Different spin label lipid analogs exhibit different selectivity for the whole AChR protein and its transmembrane domains. In all cases labeled residues were found to lie in a shallow position. For M4 segments, this is compatible with a linear ␣-helical structure, but not so for M1, for which "classical" models locate Cys residues at the center of the hydrophobic stretch. The transmembrane topography of M1 can be rationalized on the basis of the presence of a substantial amount of non-helical structure, and/or of kinks attributable to the occurrence of the evolutionarily conserved proline residues. The latter is a striking feature of M1 in the AChR and all members of the rapid ligand-gated ion channel superfamily.The muscle and electric organ nicotinic acetylcholine receptor (AChR) 1 is a pentameric integral transmembrane protein of homologous ␣ 2 ␤␥␦ subunits. The AChR belongs to a superfamily of ligand-gated ion channels, together with the glycine receptor, a subtype of the serotonin receptor (5-HT 3 ), and the GABA A receptor (1-4). Each AChR subunit contains a relatively large amino-terminal extracellular domain of ϳ200 amino acids followed by four hydrophobic domains of 20 -30 amino acids in length (M1-M4) connected by hydrophilic loops of varying length and ending with a very short extracellular carboxyl terminus (reviewed in Ref. 5).Although the exact topology of the AChR relative to the membrane has not yet been determined unambiguously, it is usually accepted that the four hydrophobic segments M1-M4 correspond to transmembrane (TM) domains (6 -7). There is still contradictory evidence on their secondary structure. The original postulation of a four-helix bundle with an all-helical secondary structure (8) has been challenged by the results of cryoelectron microscopy of frozen AChR tubules (9 -10) and computer-aided molecular modeling indicating that the dimensions of the AChR TM region are not compatible with a pentameric four-helix bundle (11). Site-directed mutagenesis data combined with patch clamp electrophysiology, and results from photoaffinity labeling with noncompetitive channel blockers, support the notion that the M2 domain lines the walls of the ion channel proper and are indicative of ␣-helical periodicity in the residues exposed ...

alpha-Conotoxins (alpha-CgTxs) are a family of Cys-enriched peptides found in several marine snails from the genus Conus. These small peptides behave pharmacologically as competitive antagonists of the nicotinic acetylcholine receptor (AChR). The data indicate that (1) alpha-CgTxs are able to discriminate between muscle- and neuronal-type AChRs and even among distinct AChR subtypes; (2) the binding sites for alpha-CgTxs are located, like other cholinergic ligands, at the interface of alpha and non-alpha subunits (gamma, delta, and epsilon for the muscle-type AChR, and beta for several neuronal-type AChRs); (3) some alpha-CgTxs differentiate the high- from the low-affinity binding site found on either alpha/non-alpha subunit interface; and that (4) specific residues in the cholinergic binding site are energetically coupled with their corresponding pairs in the toxin stabilizing the alpha-CgTx-AChR complex. The alpha-CgTxs have proven to be excellent probes for studying the structure and function of the AChR family.