It has been shown recently that in pyramidal neurons of the rat prefrontal cortex, low-threshold Nav1.6 and high-threshold NaA1.2 channels preferentially distribute at the distal and proximal axon initial segment (AIS), responsible for action potential initiation and backpropagation, respectively.However, this scenario is confined to situations where action potentials initiate from the resting membrane potential (Vm), given that in physiological conditions, somatic Vm of pyramidal cells always fluctuate under synaptic barrages.Here we report that depolarization of somatic Vm, during either current injection or "Up" states in slow oscillation, substantially inactivates AIS sodium channels (NaChs) and, strikingly, reduces the availability of inactivation-prone Nav 1.6 in distal AIS to a level lower than 10%.Further modeling study captures this observation, and suggests that a ~30 times higher density of AIS NaChs compared with the soma and a ~14 mV shift in voltage dependence of Nav1.6 and Nav1.2 found previously might be set so in order to guarantee a stable action potential initiation site within a physiological range of somatic Vm, especially during severe inactivation of NaChs.Therefore, our findings might resolve the discrepancy between different previous studies on axonal NaChs, by arguing that the dynamic collaboration of NaChs should be taken into account when comparing the anatomical and the functional results.