Pyrenesulfonyl azide: a marker of acetylcholine receptor subunits in contact with membrane hydrophobic environment.

A lipophilic photolabel, [3H] pyrenesulfonyl azide, has been synthesized and employed to detect which portions of the acetylcholine receptor (AcChR) molecule may be in contact with the hydrophobic environment of Torpedo californica electroplax membranes. The probe preferentially partitions into the hydrophobic regions of membrane lipids or Triton X100 micelles of detergent-solubilized membranes. When irradiated by UV light, the azide generates a nitrene compound which binds covalerttly, and preferentially, to membrane proteins. In Triton X-100 solubilized AcChR, the 40000 and 48 000 molecular weight subunits are preferentially labeled, whereas in situ membrane labeling produces incorporation of the radioactive photoproduct in the 48 000 and T h e nicotinic acetylcholine receptor (AcChR)' is a protein localized a t some synaptic junctions and in the electric organ of certain fishes. AcChR isolated from Torpedo californica has a molecular weight of 270000 (Martinez-Carrion et al., 1975a) and contains four different polypeptide chains with apparent molecular weights of 40 000, 50 000, 60 000, and 65000 (Chang & Bock, 1977; Flanagan et al., 1976; Hucho et al., 1976; Karlin et al., 1975; Nickel & Potter, 1973; Raftery et al., 1976). There is increasing evidence that the 40000 subunit is implicated in processes involving ligand interactions (Karlin et al., 1975; Hucho et al., 1976; Witzemann & Raftery, 1977, 1978; Hsu & Raftery, 1979), whereas there is very little knowledge about the functional role(s) or topography of the other subunits within the postsynaptic membrane. Hydrophobic probes suitable for labeling membrane components, such as those containing photogenerated arylnitrenes or carbenes, have previously been designed, with mixed results. These probes appear to bind with varying efficiency to both protein and lipid components of membranes (Nieva-Gomez & Gennis, 1977; Bayley & Knowles, 1978a,b; Klip & Gitler, 1974; Bercovici & Gitler, 1978). Furthermore, very little is known about the selectivity of the photogenerated probes; arylnitrenes label both proteins and lipids (Klip & Gitler, 1974; Bercovici & Gitler, 1978), while carbenes show great promise as labels for both saturated and unsaturated fatty acids of lipid bilayers (Bayley & Knowles, 1978b). On the other hand, preparation of water-soluble photoaffinity labels for the binding sites of several receptors, including AcChR, has achieved greater success (Levy et al., 1977; Witzemann & Raftery, 1977, 1978; Bregman et ai., 1978; Forbush et al., 1978). We have previously reported (Sator et al., 1979) the advantages of pyrene as an adequate fluorescent probe that can be introduced into the lipid phase of AcChR-rich membranes. In this paper, we describe the synthesis and properties of a pyrene derivative as a photoprobe, suitable to enter the hydrophobic regions of the bilayer and, after photoactivation, be able to link to the polypeptide chains of AcChR presumably From the Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298. Receiued Ocrober 25, 1978. This work was supported by Grant BNS 77-24715 of the National Science Foundation. 0006-2960/79/0418-1200$01 .OO/O 55 000 subunits isolated from the membrane. In both solubilized and membrane-bound receptor, the 68 000 molecular weight subunit is poorly labeled. The results suggest that in the membrane environment the 48 000 and 55 000 molecular weight subunits have a pronounced exposure to the membrane lipids, whereas the 68 000 subunit is protected from the label, possibly being partially enveloped by the other polypeptide chains from AcChR. Variations in labeling of receptor subunits in Triton X-100 and native membrane surroundings indicate that in these two environments there is an unequal accessibility of the photolabel probe to various regions of the receptor. in contact with the lipid environment of the membrane fragments. Materials and Methods BSA, crystallized and lyophilized, was from Sigma Chemical Co., as was sodium azide; Carb and spectrograde acetone were from Aldrich Chemical Co. Protein standards for the determination of molecular weights by NaDodS04 gel electrophoresis were from Boehringer. Torpedo californica electroplax was purchased at Pacific Biomarines Supply Co. and pyreneI-sulfonyl chloride was from Molecular Probes (Roseville, MN) . a-Bgt was purified from Bungarus multicinctus venom (Sigma Chemical Co.) following published procedures (Clark et al., 1972) and the ~ ~ [ ' * ~ 1 ] B g t was prepared by iodination by the solid state lactoperoxidase method (David & Reisfield, 1974). Cobratoxin was purified from Naja naja siamensis venom (Miami Serpentarium) by the method of Ong & Brady (1974). Synthesis and Purification of Radioactiue PySA. To 0.4 g of pyrene-I-sulfonyl chloride dissolved in 10 mL of tetrahydrofuran, sodium azide (saturated solution in water) in 10 molar excess over pyrenesulfonyl chloride was added and the mixture stirred overnight. Subsequently, 15 mL of water was added and the precipitate was collected and treated again with sodium azide, under the same conditions, for another 6 h. The crude product was recrystallized from benzene, giving a total yield of about 80% in weight. The recrystallized product was further purified by TLC on silica gel G, using chloroform as a solvent (Rt0.74), and then eluted with chloroform in a sintered-glass funnel giving a final yield of 65%. Elemental analysis of the synthetic material was determined. Anal. Calcd for C,6H9N,02S: C, 63.41; €3, 2.97; N, 13.87; S, 10.58. Found: C, 63.29; H, 2.95; N , 13.28; S, 10.42. IR spectra in KBr pellets show the presence of the band characteristic of sulfonyl azides (-2100 cm-I). The purified material shows no presence of impurities, as revealed by TLC using chloroform and mixtures of chloroform/methanol (1 0: 1, 5: 1, and 1: 1, by vol) or n-hexaneldiethyl etherlacetic acid/ . ' Abbreviations used: AcChR, acetylcholine receptor; BSA, bovine serum albumin; Carb, carbamoylcholine; a-Bgt, a-bungarotoxin; PySA, pyrenesulfonyl azide; TLC, thin-layer chromatography; PhCH2S02F, phenylmethanesulfonyl fluoride; NaDodSO.,, sodium dodecyl sulfate; DAPA, bis(3-aminopyridinium)-l ,lO-decane azide, ETA, ethidium azide.