The presynaptic action potential (AP) is required to drive calcium influx into nerve terminals, resulting in neurotransmitter release. Accordingly, the AP waveform is crucial in determining the timing and strength of synaptic transmission. The calyx of Held nerve terminals of rats of either sex showed minimum changes in AP waveform during high-frequency AP firing. We found that the stability of the calyceal AP waveform requires KCNQ (KV7) K+ channel activation during high-frequency spiking activity. High-frequency presynaptic spikes gradually led to accumulation of KCNQ channels in open states which kept interspike membrane potential sufficiently negative to maintain Na+ channel availability. Blocking KCNQ channels during stimulus trains led to inactivation of presynaptic Na+, and to a lesser extent KV1 channels, thereby reducing the AP amplitude and broadening AP duration. Moreover, blocking KCNQ channels disrupted the stable calcium influx and glutamate release required for reliable synaptic transmission...