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

Calcium ions (Ca 2+ ) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca 2+ imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca 2+ ] i ) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca 2+ ] i changes were sensitive to the specific voltage-gated sodium (Na V ) channel blocker tetrodotoxin. Consistent with the conjecture that Ca 2+ 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 V 1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca 2+ ] i rise. Finally, computational simulations predicted that axonal [Ca 2+ ] i transients reflect a 0.4% Ca 2+ conductivity of Na V channels. The findings indicate that Ca 2+ permeation through Na V channels provides a submillisecond rapid entry route in Na V -enriched domains of mammalian axons. Nerve cells communicate using tiny electrical impulses called action potentials. Special proteins termed ion channels produce these electric signals by allowing specific charged particles, or ions, to pass in or out of cells across its membrane. When a nerve cell ‘fires’ an action potential, specific ion channels briefly open to let in a surge of positively charged ions which electrify the cell. Action potentials begin in the same place in each nerve cell, at an area called the axon initial segment. The large number of sodium channels at this site kick-start the influx of positively charged sodium ions ensuring that every action potential starts from the same place. Previous research has shown that, when action potentials begin, the concentration of calcium ions at the axon initial segment also increases, but it was not clear which ion channels were responsible for this entry of calcium. Channels that are selective for calcium ions are the prime candidates for this process. However, research in squid nerve cells gave rise to an unexpected idea by suggesting that sodium channels may not exclusively let in sodium but also allow some calcium ions to pass through. Hanemaaijer, Popovic et al. therefore wanted to test the routes that calcium ions take and see whether the sodium channels in mammalian nerve cells are also permeable to calcium. Experiments using flu