Objectives: 

This work was aimed to determine the inhibitory effects of diethylamine (DEA), which resembles the hydrophilic moiety of the lidocaine molecule, on muscle-type nicotinic acetylcholine receptors (nAChRs), since lidocaine, partially protonated at physiological pH, has multiple inhibitory actions on nAChRs (Alberola-Die et al., 2011; J. Neurochem. 117:1009-1019).

Materials: 

Purified Torpedo nAChRs were transplanted to Xenopus oocytes and currents elicited by ACh (IAChs), either alone or co-applied with lidocaine or DEA, were recorded at different membrane potentials. Besides, giant proteoliposomes bearing nAChRs were used to record single-channel IAChs (excised patch, inside-out configuration), in the presence or absence of DEA. Docking simulations were also carried out to screen the possible binding sites of these molecules to the nAChR.

Results: 

DEA reversibly blocked IACh, in a dose-dependent way (IC50 close to 70 microM; Hill coefficient of 1) without affecting the rate of IACh desensitization. IACh inhibition by DEA was only present at negative potentials, suggesting an open-channel blockade. The electrical distance of the DEA binding into the channel pore was similar to that reported for lidocaine (d=0.3). Furthermore, DEA blocked nAChRs in the resting (closed) state, indicating additional binding sites for this molecule outside the pore. Lidocaine, when applied at low doses (below the IC50), showed similar effects on nAChRs to those evoked by DEA. Single-channel experiments confirmed that DEA blocked nAChRs by decreasing the channel conductance and the open channel probability.

Conclusions: 

Functional data and docking assays indicate that the hydrophilic region of lidocaine and DEA accounts for the open-channel blockade of nAChRs, binding at roughly 30% of the channel depth. Besides, lidocaine and DEA interacts with residues located outside the ion channel.

This work was supported by grant CSD2008-00005 from the MEC (Spain).