Interaction of midazolam with the nicotinic acetylcholine receptor of mouse myotubes

The effects of midazolam on the peripheral embryonic; nicotinergic acetylcholine receptor (nAChR) of mouse myotubes were studied to elucidate the mechanism of its effect on neuromuscular transmission. Standard patch clamp techniques on outside-out patches were used. Pulses of 10(-4) M acetylcholine (ACh) applied by a liquid filament switch technique elicited macroscopic channel currents with a peak current amplitude of approximately 40 pA within < 1 ms. The current decayed with a time constant of 30-100 ms due to desensitization. When midazolam was added in stepwise increased concentrations (10(-7) M to 7 x 10(-4) M) to the pulses, the current decay became bi-exponential, and a concentration-dependent decrease of the fast component of decay was observed. The current amplitude, however, was reduced slightly, and only at high concentrations of midazolam. This may indicate that midazolam binds to the open channel to cause the block. The rate constant of block (b(+1)) was found to be 1.8 x 10(6) M/s. Recovery experiments revealed a rate of unblocking (b(-1)) of approximately 2 x 10(-1) s(-1). After preincubation of the patches with midazolam, a substantial reduction of the current amplitude was seen at very low midazolam concentrations (< 10(-7) M), which suggests an additional closed channel block with a K-d of approximately 10(-6) M. This closed channel block may be responsible for the muscle-relaxing effects of midazolam.