The Binding of Tetrodotoxin and Alpha-bungarotoxin to Normal and Denervated Mammalian Muscle

1. The binding of [(3)H]tetrodotoxin and [(125)I]iodo-alpha-bungarotoxin to innervated and denervated rat diaphragm muscle has been measured.2. A saturable component of tetrodotoxin binding, which was inhibited by saxitoxin, was detected in addition to considerable non-saturable binding. The saturable component had an equilibrium constant of K = 6.1 nM (limits 4.7-7.8 nM) and binding capacity M = 2.5 f-mole/mg wet wt. (limits 2.1-2.8 f-mole/mg).3. If the saturable component consisted of one-to-one binding of tetrodotoxin to sodium channels, the density of sodium channels would be about 21/mum(2) of surface membrane, a figure similar to that found in other excitable membranes.4. After denervation the specific tetrodotoxin binding, as measured by the ratio M/K, fell by a factor of 2.8. This change appeared to be due to a fall in binding capacity rather than a decrease in affinity.5. After denervation the maximum rate of rise of the action potential fell by 27% and became partially resistant to tetrodotoxin. The maximum rate of rise was at first reduced by tetrodotoxin in similar concentrations to those affecting normal muscle, but even large concentrations which completely blocked normal muscle only reduced the maximum rate of rise by a factor of about 2.6. Detubulation with glycerol did not appreciably affect the tetrodotoxin sensitivity of normal or denervated muscle.7. Tetrodotoxin resistance was not observed after denervation of the frog sartorius muscle.8. [(125)I]iodo-alpha-bungarotoxin binding amounted to 3.8 +/- 0.7 f-mole/mg in innervated muscle and 44.5 +/- 2.1 f-mole/mg in denervated muscle. Most of the uptake was inhibitable by (+)-tubocurarine.9. The increase in the labelled bungarotoxin binding is much larger than the specific tetrodotoxin binding of innervated muscle, which renders implausible the possibility that the acetylcholine receptors which appear after denervation are related to the tetrodotoxin resistant-sodium channels.