Influence of chloride ions on changes in membrane potential during prolonged application of carbachol to frog skeletal muscle.

1. Micro-electrodes were used to follow changes in the membrane potential at the end-plate region of single fibres in narrow strips of frog skeletal muscle exposed to carbachol applied in continuously flowing Ringer solution containing tetrodotoxin (200 nM) and neostigmine (3 muM).2. The depolarizations elicited by carbachol (5-20 muM) usually developed in two phases, the first of which was generally complete within 30 s whereas several min were required for the second.3. Repolarization after carbachol also occurred in two phases, the second of which outlasted the time needed to clear the bath, and varied with the magnitude and duration of the depolarization which carbachol had caused.4. These findings could best be explained in terms of the consequences of net entry of chloride ions into the fibre during the depolarization caused by carbachol. This hypothesis is supported by three lines of evidence:(a) Replacement of the chloride content of the Ringer solution by the less permeant anion isethionate abolished the slow phases of the carbachol response.(b) Reduction of chloride permeability (by lowering pH) caused rapid repolarization during the recovery period after carbachol.(c) When the membrane potential was clamped at the resting level throughout the action of carbachol, so avoiding chloride redistribution, the clamping current records did not show the slow phases attributed to chloride movement.5. Chloride redistribution contributes to the gradual spread of depolarization during prolonged applications of depolarizing agents to skeletal muscle. It also complicates the interpretation of the dose-response relationship, and may make it more difficult to assess the extent to which the receptors become desensitized during the action of agonists applied in the bath.