It is now generally accepted that most pharmacological agents act at specific molecular sites on the cell membrane, called receptors. Only cells which possess the appropriate receptor will respond to a particular agent. In accordance with this view, we would expect to find specific acetylcholine receptors on the postsynaptic membrane at the neuromuscular junction.
The substance a-bungarotoxin, a polypeptide found in the venom of a Formosan snake, causes neuromuscular block by binding tightly to the acetylcholine receptors. Using radioactive toxin (made by acetylating the toxin with 3H-acetic anhydride) it is a simple matter to show by autoradiography that the toxin rapidly becomes attached to the postsynaptic membrane at the end-plate regions. By counting the grains of silver produced in the autoradiograph, it is then possible to count the number of toxin molecules that have been bound and from this to estimate the number of receptors present. The results suggest that there are about 3 X107 binding sites per end-plate in mammals, corresponding to an average density in the region of 104 sites per fxm2.
Since combination of acetylcholine with the receptors causes an increase in membrane permeability to sodium and potassium ions, it seems very likely that each receptor is closely associated with an ion channel through which this ionic flow can occur. It would normally be closed and would open for a short time when acetylcholine combines with the receptor.
Direct evidence for this view was provided in experiments by E. Neher and B. Sakmann, for which they developed the patch-clamp technique (see Fig. 4.17). They used frog muscle fibres whose motor nerve supply had been cut some time previously. Following this procedure the whole surface of the fibre becomes sensitive to acetylcholine and contains a low density of acetylcholine receptors. The polished tip of a microelectrode can then be pushed against the fibre membrane, so that the current flow through a small patch of membrane containing only a few receptors can be measured.
Neher and Sakmann found that, when the patch electrode contains acetylcholine, individual channels each produce a square pulse of current lasting up to a few milliseconds. The durations of successive current pulses were variable, but their amplitudes were constant, as is shown in Fig. 7.9. This suggests that the channel is either open or shut, and that it can only open when it combines with acetylcholine. Probably two molecules of acetylcholine need to combine with each receptor in order to open the channel. The end-plate potential is thus produced when a large number of channels open more or less at the same time.
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