Molecular Pharmacology, Vol 20, 345-355, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

The Ionic Channel of the Acetylcholine Receptor

Regulation by Sites Outside and Inside the Cell Membrane Which Are Sensitive to Quaternary Ligands

¹ Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201, Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029, and Laboratory of Bioorganic Chemistry, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20205

The actions of several quaternary molecules on the end plate region of the frog sartorius muscle were studied using bath application or intracellular injection. Tetraethylammonium (TEA), atropine methyl bromide, phencyclidine methiodide (PCP methiodide), piperocaine methiodide, and N-methyl piperidine methiodide were injected into the sarcoplasm just beneath the postjunctional membrane and 250-350 µm away from the end plate region. The ability of these agents to depress potassium conductance and prolong the muscle action potential was used as a measure of the efficacy of intracellular drug administration. External application of TEA (50-1000 µM) decreased the peak amplitude of the end plate current (EPC) and its time constant of decay (_EPC), but this agent and atropine methyl bromide were ineffective when injected internally. PCP methiodide (3-30 µM) and piperocaine methiodide (10-60 µM) had a potent action on EPCs and spontaneous miniature end plate currents (MEPCs) when applied to either side of the membrane. Both agents caused nonlinearity of the peak amplitude and a shortened channel lifetime in spite of the fact that they sense only 6% of the membrane potential at their rate-limiting energy barrier. Internal application of PCP methiodide caused significant depression of the EPC and MEPC peak amplitude and simultaneous shortening of MEPC decay time constant. The decay time constant of the EPC and MEPC in the presence of PCP methiodide and piperocaine methiodide was shorter at less negative (i.e., -60 mV) than at more negative (i.e., -100 mV) membrane potentials. Similar results were obtained with internal applications of piperocaine methiodide. N-methyl piperidine methiodide, a quaternary contaminant of PCP methiodide, did not display any effect when it was applied inside the cell. The results indicate that sites controlling the ionic channel of the acetylcholine receptor from the external surface may be significantly different from sites on the internal surface, even though both sites do interact with certain quaternary amines. These findings further indicate that the ionic channel is asymmetrical with the selectivity gate located most likely at the intracellular region of the channel. Finally, it is suggested that the decay time constant of the ionic currents in the presence of tertiary agents may be an average, voltage-dependent result of the actions of the agent inside and outside the cell.

Note:
ACKNOWLEDGMENTS The authors are grateful to Professor David Triggle for the gift of PCP methiodide. We are most indebted to Dr. Harel Weinstein and Dr. Saul Mayaani for the most valuable suggestions for and criticisms of the manuscript. We are also grateful to Dr. Daniel Weinreich for the technical suggestions regarding intracellular release of drugs. The expert computer analysis and general technical assistance of Ms. Mabel Zelle are greatly appreciated.