Molecular Pharmacology, Vol 16, 909-921, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Voltage- and Time-dependent Actions of Piperocaine on the Ion Channel of the Acetylcholine Receptor

¹ Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201

The effects of the local anesthetic piperocaine were investigated on the endplate current (EPC) of frog sartorius muscles and on the binding of ligands to the acetylcholine (ACh) receptor and its ion channel in membranes from the electric organ of Torpedo ocellata. Piperocaine (10-100 µM) did not prevent action potential activity in nerve or muscle. However, these concentrations of piperocaine depressed reversibly the peak amplitude of EPCs in a dose-dependent manner without altering the EPC reversal potential. The current-voltage relationship obtained with short conditioning voltage durations preceding the EPC remained approximately linear at the piperocaine concentrations used. When the time during which the membrane potential was maintained preceding the EPC was lengthened from 10 to 500 msec in presence of piperocaine, the current-voltage relationship became markedly nonlinear, thus suggesting that there was more binding of the drug to the ACh-receptor ion channel complex. Both drug concentration and increasingly negative membrane potential augmented this time-dependent effect. At negative membrane potentials piperocaine also reversibly accelerated the rise and decay times of the EPC, while the EPC falling phase remained a single exponential function of time. The relationship between log of EPC decay time constant () and membrane potential was linear in presence of piperocaine, and the slope progressively decreased and reversed its direction as piperocaine concentration was increased, with the maximum observed acceleration of being at 75 µM of drug. The effect of piperocaine on was voltage dependent but time independent. Piperocaine inhibited competitively [³H]perhydrohistrionicotoxin binding to the electric organ membranes, with an inhibition constant (K_i) of 0.44 µM; and noncompetitively [³H]ACh binding to its ACh-receptor with a K_i of 12.0 µM. These findings suggest that piperocaine has at least two separate actions at the ACh-receptorion channel complex. One is binding to open channels which causes concentration and voltage-dependent alteration of EPC time course and decreased EPC amplitude and voltage sensitivity of the EPC falling phase. Another is binding to a less well-defined site on the ACh receptor ion channel complex, an action that leads to a further depression of the peak amplitude of the EPC which is concentration, voltage and also time-dependent.

Note:
ACKNOWLEDGMENT We are grateful to Drs. B. Witkop and J. Daly of NIH for supplying us with the [³H]H₁₂-HTX used in the present study. We are most indebted to Ms. Mabel A. Zelle for all the computer analysis.