374 Biochirnica et Biophvsica A cta, 798 (1984) 374- 381 Elsevier BBA 21719 CONVERSION OF ACETYLCHOLINE RECEPTOR DIMERS TO MONOMERS UPON DEPLETION OF NON-RECEPTOR PERIPHERAL PROTEINS MANUEL CRIADO * and FRANCISCO J. BARRANTES ** Max - Planck - Institut fur biophysikalische Chemie, Postfach 2841, 3400 G6ttingen - Nikolausberg (F. R. G.) (Received August 9th, 1983) Key words: A cetylcholine receptor," Dimer-monomer conversion," (T. marmorata) The influence of treatments for extracting non-receptor peripheral proteins on the oligomeric states of the acetylcholine receptor has been studied in receptor-rich membranes from Torpedo marmorat~ Conventional alkaline treatment of non-alkylated membranes resulted in the extraction of peripheral proteins (30% of total membrane proteins). Concomitantly, partial conversion of the dimer into the monomer was observed in the absence of exogenous reduction. Alkaline extraction at high ionic strength resulted in a marked decrease in protein solubilization, and no conversion of the dimer to the monomer occurred. Alkaline treatment extracted only one half of the peripheral proteins (15% of total protein) from membranes previously alkylated with N-ethyimaleimide or iodoacetamide, or oxidized by sodium periodate. Com;ersion of dimer to monomer was totally prevented by these treatments. Similar results were obtained by treatment of the membranes with lithium 3,5-diiodosalicylate. The above effects of alkaline extraction on the acetyicholine receptor can be interpreted in the context of two mutually non-exclusive mechanisms: (a) some of the peripheral proteins may directly participate in the thiol-dependent receptor aggregational states. Their extraction destroys this dynamic control. (b) Extraction of peripheral proteins destabilizes the receptor and makes it more susceptible to inter or intramolecular sulfhydryl-disulfide exchange, leading to the endogenous reduction of a proportion of the dimers. Introduction The acetylcholine receptor is an intrinsic mem- brane protein involved in the regulation of the postsynaptic membrane permeability to cations (for recent reviews, see Refs. 1,2). In purified membranes of electric organ of Torpedo this protein is composed of five subunits, two of which carry the binding site for acetylcho- * To whom correspondence should be addressed (present address): The Salk Institute, P.O. Box 85800, San Diego, CA 92138, U.S.A. ** Present address: Institutode Bioquimica, 8000 Bahia Blanca, Argentina. Abbreviation: PMSF, phenylmethylsulfonyl fluoride. line (a-chains, M r 38000) and the other three of molecular weights determined by SDS-gel electro- phoresis) do not as yet have a well defined func- tion. The involvement of the &chain in the bind- ing of non-competitive blockers has been sug- gested [3,4]. This polypeptide is also responsible for the covalent association of the acetylcholine receptor into dimers through a disulfide bond [5-7]. A comprehensive analysis of the transmem- brane orientation and tryptic products of the &subunit has been published [8]. The minimal functional unit of the acetylcholine receptor ap- pears to be the monomer, at least in terms of 22Na+ integrated flux [9] and single-channel behavior in reconstituted systems [10]. The lack of any detected difference between monomers and dimers does not mean that these forms are really 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V.