Modifications of Single Acetylcholine-activated Channels BC3H-1 Cells in Effects of Trimethyloxonium and pH

We have examined the effects o f chemical modification with tr imethyloxonium (TMO) and changes in external p H on the proper t ies o f acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by ~60%. G~ seal patch-clamp recordings o f single ACh channel currents showed that the reduct ion in ACh sensitivity is due to alterations in both the current-carrying and the kinetic proper t ies o f the channels. Unde r all our experimental conditions, i.e., in external solutions o f normal o r low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh singlechannel conductance to 70-90% o f normal. The effects o f TMO on channel kinetics were dependen t on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by ~25%. A similar reduct ion in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics o f modif ied channels. In low ionic strength solutions normal ACh channel open times were maximal at p H 6.7 and decreased by threeto fourfold at both acid and alkaline pH. TMO modification removed the p H dependence o f channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally t i tratable groups on the external surface of ACh channels that help to determine both the gating and permeabili ty proper ties o f ACh channels. Address reprint requests to Dr. Pamela Pappone, Department of Animal Physiology, University of California, Davis, CA 95616. Dr. Barchfeld's current address is Chiron Corporation, Q3-14, 4560 Horton Street, Emeryville, CA 94608-2916. J. GEN. PHYSIOL. ~) The Rockefeller University Press 9 0022-1295/90/07/0001/22 $2.00 1 Volume 96 July 1990 1-22 on A ril 6, 2016 jgp.rress.org D ow nladed fom Published July 1, 1990