Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions

Abstract

The distribution and quantitation of (125)I-α-bungarotoxin (α- BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetantic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, α-BTX was bound to approximately 90 percent of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of α-BTX relative to this membrane was determined to be 46,000 +/- 27 percent sites/μm(2). By testing various hypothetical models the distribution of developed grains was found to be consistent with the hypothesis that the main acetylcholine-receptive surface coincides with the electron-dense, thickened portion of the junctional fold membrane situated at the juxtaneuronal region of the folds and dipping approximately 2,200 A be approximately 30,500 +/- 27 percent sites/μm(2) of this postsynaptic dense membrane. There was a sharp gradient in α-BTX binding extrajunctionally, with the concentration decreasing to approximately 4 percent of the subsynaptic value within 1μm from the edge of an axonal terminal (bouton) to <1 percent within 3μm and to <0.2 percent beyond 7 μm from that terminal. Below 4,000 A (i.e. half-way from the top of the junctional folds) the concentration of α-BTX was also about 3 percent of the peak subsynaptic value. The binding density at the bottom of the junctional folds is this comparable to extrajunctional sarcolemma at equal distance from a nerve terminal. The molecular organization at the neuromuscular junction relative to its function is discussed.