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Pharmacology Institute, College of Medicine, National Taiwan University, Taipei, Taiwan 100, Republic of China

1. The recovery of contractile responses and appearance of new -bungarotoxin-binding sites were studied in the baby chick biventer cervicis and the rat diaphragm muscles after saturating the existing acetylcholine (ACh) receptors (AChR) with -bungarotoxin in vitro.

2. Washout of -bungarotoxin restored gradually the response to exogenous ACh attaining about 30% recovery in 3 hr either in the chick muscle or in the denervated rat diaphragm. No recovery was obtained, however, for the response to nerve stimulation.

3. The recovery of ACh-response was abolished by decreasing the bath temperature to 9° C during the washout of the toxin whereas the recovery was not reduced in the presence of cycloheximide.

4. The half-life of [³H]acetyl -bungarotoxin bound specifically on the existing AChRs, junctional and extrajunctional receptors combined, was 16 hr in the chick muscle. That on the extrajunctional AChR was estimated to be 8 hr.

5. New toxin-binding sites were found to be incorporated on the membrane of extrajunctional site rapidly after treatment with -bungarotoxin in the chick and the denervated rat muscles along the muscle fibres but not in the innervated rat diaphragm. Treatment with (+)-tubocurarine, ACh or decamethonium did not cause an appreciable increase of the toxin-binding sites.

6. The appearance of new binding sites was progressive during 5 hr at a rate of 24 sites/µm².hr in the chick muscle and 42 sites/µm².hr in the rat diaphragm denervated for 7 days. The existing extrajunctional AChR were about 50/µm² and 192/µm², respectively.

7. ACh effectively antagonized the binding of -bungarotoxin with the new sites whereas (+)-tubocurarine was less effective than its effect on the existing AChR.

8. The new toxin-binding sites appeared to have a reduced capacity to evoke ACh response.

9. The incorporation of new binding sites was reduced by lowering of the temperature, treatment with dinitrophenol, high K⁺, high Ca²⁺ and by the stimulation of either nerve or muscle. Cycloheximide, ACh, decrease of [Na⁺]_o and increase of [Mg²⁺]_o were without effect.

10. It is suggested that binding of the extrajunctional AChRs with -bungarotoxin cause a change of membrane architecture and trigger the incorporation of cytoplasmic AChR-precursor or hidden AChR into the membrane.