A morphological study of the cholinergic receptor protein from Torpedo marmorata in its membrane environment and in its detergent-extracted purified form.

Abstract

ABSTRACT The highly specialized structural features of the postsynaptic membrane of Torpedo mar-morata electric organ have been studied by freeze-fracturing, freeze-etching and negative staining applied both to intact electroplaques and to isolated purified excitable membrane fragments. The external surface of the postsynaptic membrane, revealed by deep-etching and negative staining, is characterized by the presence of geometrically packed arrays of repeating particles having a subunit structure and a central pit. The isolated receptor protein molecules (molecular weight : 300 000) show structural features identical to those characteristic of the repeating particles visualized on the external surface of the postsynaptic membrane. Freeze-fracture cleaves the postsynaptic membrane into 2 asymmetric halves, showing that numerous identical particulate entities are more strongly anchored to the cytoplasmic side of the bilayer. On the other hand, geometrically packed arrays of pits or depressions found on the external fracture face suggest that the receptor protein extends across the outer leaflet and is exposed to the external surface facing the synaptic cleft. The homogeneous size-distribution of the particulate entities associated with the postsynaptic membrane element is consistent with the finding that the receptor protein accounts for at least 40% of the total protein of purified excitable membrane fractions. These results strongly favour the assumption that the particulate entities represent the receptor protein, which forms oligomeric complexes either of identical polypeptides or quasi-equivalently related proteins. Thus, the cholinergic receptor oligomers forming a bidimensional lattice of repeating particles are exposed both to the synaptic cleft and to the cytoplasmic environment. It is proposed that the regulating properties of the excitable membrane depend upon oligomeric or polymeric associations of receptor proteins spanning the bilayer.