Alpha-bungarotoxin Binding Sites Research Articles

Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity.

1. A linear relation, with a slope of 0-9 +/- 0-2 on a log-log plot, was obtained between acetylcholine (ACh) sensitivity and alpha-bungarotoxin (alpha-BTX) binding site density in developing L6 and rat primary myotubes. ACh sensitivity was defined as g/Qn where g is conductance, Q is ACh charge and n is the Hill coefficient. Experimentally we found n approximately 1-7 for our myotubes, which is similar in value to that reported for adult systems. 2. The linear relationship is compatible with an organization whereby each ion channel is always complexed with a fixed number of ACh receptors such that the dose-response characteristics of each such complex are independent of average ACh receptor density. 3. Light microscope autoradiography showed that the alpha-bungarotoxin binding sites on L6 myotubes are uniformly distributed over the surface, while primary rat myotubes exhibit gradients and hot spots. Electron microscope autoradiography indicated that about 70% of the [125I]alpha-bungarotoxin label was on the surface of the myotubes. The alpha-bungarotoxin site density, after subtracting myoblast background, varied from 5 to 400 sites/micrometer2 on different L6 myotubes, and from 54 to 900 sites/micrometer2 on primary rat myotubes, with occasional hot spots of 3000-4000 sites/micrometer2. The conductance sensitivities varied from 10(-4) to 2 X 10(-2) Momega-1/nC1-7.

The distribution of alpha-bungarotoxin binding sites of mammalian skeletal muscle developing in vivo.

1. The distribution of alpha-bungarotoxin binding sites on embryonic and neonatal rat skeletal muscle fibres was determined by autoradiography. Most of the bungarotoxin binding could be inhibited by curare. This observation, together with the spatial distribution of toxin-binding sites, indicates that the distribution of bound toxin reflects that of acetylcholine (ACh) receptors on these developing muscle cells.2. At 15 days of embryogenesis, muscle fibres showed an essentially uniform distribution of receptors. By 16 days, many fibres showed an accumulation of receptors in their mid-region. This accumulation was at the same location as histochemically demonstrated cholinesterase activity.3. At 16 days ACh receptors were distributed over the entire length of the fibres, with a gradient of increasing density as the accumulation was appoached. The density of toxin binding sites in the accumulation was greater than the general level on 15 day cells, suggesting that the high junctional density does not develop solely by the loss of extrajunctional receptors.4. The accumulations of ACh receptors became more pronounced and circumscribed with embryonic development, and after birth the extent of the localizations appeared to follow the size of the neuromuscular junction. The extrajunctional receptor density decreased with development, and by 1 week after birth was undetectable by the methods used.5. The results suggest that the high junctional receptor density found on adult, innervated skeletal muscle fibres develops after the formation of the neuromuscular junction.

Acetylcholine receptor turnover in membranes of developing muscle fibers.

[125I mono-iodo-alpha-bungarotoxin is used as a specific marker in a description of acetylcholine receptor metabolism. It is concluded that acetylcholine receptors in the surface membranes of chick and rat myotubes developing in cell cultures have a half-life of 22-24 h. Alpha-bungarotoxin (bound to a receptor which is removed from the membrane) is degraded to monoiodotyrosine which appears in the medium. Several observations are consistent with a model in which receptors or alpha-bungarotoxin-receptor complexes are internalized and then degraded: (a) the rate of appearance of iodotyrosine does not reach its maximal rate until 90 min after alpha-bungarotoxin is bound to the surface receptors; (b) 2,4-dinitrophenol, reduced temperature, and cell disruption all inhibit the degradation process. The degradation of surface receptors is not coupled to the process by which receptors are incorporated into the membrane. Evidence suggest that receptors are incorporated into the surface membrane from a presynthesized set of receptors containing about 10% as many alpha-bungarotoxin binding sites as does the surface. Additionally, a third set of acetylcholine receptors is described containing about 30% as amny binding sites as does the surface. These "hidden" recptors are not precursors yet are not readily accessible for binding of extracellular alpha-bungarotoxin. These findings are discussed in relation to both plasma membrane biosynthesis and control of chemosensitivity in developing and denervated skeletal muscle.

Chemical Nature of Synaptic Transmission in Vertebrates

Chemical Nature of Synaptic Transmission in Vertebrates