The role of a reactive disulphide bond in the function of the acetylcholine receptor at the frog neuromuscular junction

Abstract

1. The effects of disulphide bond reduction and reoxidation on synaptic transmission in the frog neuromuscular preparation have been studied.2. The amplitudes of end-plate potentials (e.p.p.s) and miniature e.p.p.s (m.e.p.p.s) were decreased irreversibly by the reducing agent dithiothreitol (DTT). Recovery of e.p.p.s and m.e.p.p.s could be achieved, however, by subsequent reoxidation employing 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB).3. No change in e.p.p. quantal content was produced by treatment with DTT and DTNB. The reduction of m.e.p.p. frequency found in DTT was attributed mainly to our inability to distinguish small m.e.p.p.s from the background noise. However, a genuine reduction in m.e.p.p.s frequency could not be ruled out.4. The ;reactive' disulphide bond which is acted upon by DTT and DTNB could be located within a few Angstrom from the anionic site of the receptor for acetylcholine (ACh), by employing the active-site directed reagent bromoacetylcholamine (BACA).5. Reduction of the ;reactive' disulphide bond did not cause changes in the post-synaptic membrane input impedance nor was the e.p.p. reversal potential affected. Treatment with DTT and DTNB was found to modify only the conductance of the synaptic membrane.6. No synaptic effects were produced by DTT in a non-cholinergic synapse, the crab neuromuscular preparation.7. It is concluded that the receptor for ACh, besides including the well-known anionic site for binding quaternary ammonium groups, also contains a unique ;reactive' disulphide bond. This bond controls synaptic conductance but not the relative permeability of the synaptic membrane to Na(+) and K(+). It is not yet clear whether the ;reactive' bond controls the interaction of ACh with the receptor or the translation of this interaction into ionic permeability changes.