Ca²⁺ entry through Na_V channels generates submillisecond axonal Ca²⁺ signaling

Calcium ions (Ca²⁺) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca²⁺ imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca²⁺]_i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca²⁺]_i changes were sensitive to the specific voltage-gated sodium (Na_V) channel blocker tetrodotoxin. Consistent with the conjecture that Ca²⁺ enters through the Na_V channel pore, the optically resolved I_Ca in the axon initial segment overlapped with the activation kinetics of Na_V channels and heterologous expression of Na_V1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca²⁺]_i rise. Finally, computational simulations predicted that axonal [Ca²⁺]_i transients reflect a 0.4% Ca²⁺ conductivity of Na_V channels. The findings indicate that Ca²⁺ permeation through Na_V channels provides a submillisecond rapid entry route in Na_V-enriched domains of mammalian axons.