Role of disulphide bonds in burst-like activity of nicotinic acetylcholine receptor channels in rat sympathetic neurones

Abstract

1. The effects of reduction of disulphide bonds in nicotinic acetylcholine receptors (nicotinic AChRs) with dithiothreitol (DTT) were studied in rat superior cervical ganglion neurones using the patch-clamp method in whole-cell and cell-attached recording modes. 2. Dithiothreitol (1 mM) markedly reduced the ACh-induced membrane current, while the action of ACh remained reversible. Conversely, bromoacetylcholine (BrACh), if applied after the treatment with DTT, caused irreversible activation of nicotinic AChRs manifested in the appearance of a non-declined steady-state component in BrACh-induced currents accompanied by increased membrane current fluctuations. The successive reoxidation of sulphydryl groups by potassium ferricyanide (1 mM-ferricyanide) restored the response to ACh. Ferricyanide itself had a weaker inhibitory effect on the ACh-induced current, compared to the effect of DTT. 3. As a result of the action of DTT (1 mM), the spectrum of BrACh-induced current noise shifted to a higher frequency range. 4. The distributions of durations of the gaps (closed states) and the bursts (the states identified as open states after the shortest gaps were ignored) in single-channel activity of native (non-treated with DTT) nicotinic AChRs caused by ACh (30 microM) and BrACh (30 microM) were similar and both revealed four to five and two to three components for gap intervals and burst durations respectively. 5. Single-channel behaviour of reduced nicotinic AChRs was similar for both ACh and BrACh as agonists, but significantly differed from that in the native one. The first difference was the marked increase in the frequency of the appearance of long closed states of the channel that was presumably due to enhanced receptor desensitization. The second difference was an almost complete disappearance of long bursts associated with disappearance of the fastest component in gap interval distribution. 6. Mean conductance of single nicotinic AChR channels decreased by approximately 20% in the reduced receptor compared with that in the native one, for both agonists. 7. The results suggest a critical role of disulphide bonds for the functioning of native neuronal nicotinic AChRs: the disruption of disulphide bonds leads to the loss of burst-like kinetics of the nicotinic AChR ionic channel.