INTERACTION OF MIDAZOLAM WITH THE NICOTINIC ACETYLCHOLINE-RECEPTOR OFMOUSE MYOTUBES

The effects of midazolam on the peripheral embryonic; nicotinergic ace tylcholine receptor (nAChR) of mouse myotubes were studied to elucidat e 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 techniqu e 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 th e pulses, the current decay became bi-exponential, and a concentration -dependent decrease of the fast component of decay was observed. The c urrent amplitude, however, was reduced slightly, and only at high conc entrations of midazolam. This may indicate that midazolam binds to the open channel to cause the block. The rate constant of block (b(+1)) w as found to be 1.8 x 10(6) M/s. Recovery experiments revealed a rate o f unblocking (b(-1)) of approximately 2 x 10(-1) s(-1). After preincub ation of the patches with midazolam, a substantial reduction of the cu rrent 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 f or the muscle-relaxing effects of midazolam.