The effect of membrane potential on feedback inhibition of acetylcholine (ACh) release was studied using the frog neuromuscular junction. It was found that membrane potential affects the functional affinity (K i ) of the presynaptic M 2 muscarinic receptor. The K i for muscarine shifts from ϳ0.23 M (at resting potential) to ϳ8 M (at a high depolarization). Measurements of Ca 2ϩ currents in axon terminals showed that the depolarizationmediated shift in K i does not stem from

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... ent changes in Ca 2ϩ influx. Pretreatments with pertussis toxin (PTX) abolished the depolarization-dependent shift in K i ; at all depolarizations K i was the same and higher (ϳ32 M) than before PTX treatment. The inhibitory effect of muscarine on ACh release is produced by two independent mechanisms: a slow, PTX-sensitive process, which prevails at low to medium depolarizations and operates already at low muscarine concentrations, and a fast, PTX-insensitive and voltage-independent process, which requires higher muscarine concentrations. Neither of the two processes involves a reduction in Ca 2ϩ influx.