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

Abstract

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 (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+ enters through the NaV channel pore, the optically resolved ICa in the axon initial segment overlapped with the activation kinetics of NaV channels and heterologous expression of NaV1.2 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 NaV channels. The findings indicate that Ca2+ permeation through NaV channels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

Functional microstructure of CaV-mediated calcium signaling in the axon initial segment

“…Furthermore, we found that the kinetics of sodium and calcium influx were indeed identical, but that calcium influx lagged sodium influx in ways that were consistent with sodium and calcium influx occurring on the rising and falling phases of the AP, respectively. These results are consistent with studies using highspeed optical imaging, where sodium influx occurs during the rising phase of the AP (Filipis and Canepari, 2020), whereas calcium influx occurs during the falling phase of the AP in the AIS or AIS-like regions of AP initiation (Hanemaaijer et al, 2020;Pressler and Strowbridge, 2019). Furthermore, we observed consistent results at both room temperature and physiological temperatures with two different sodium-sensitive indicators and two different calcium-sensitive indicators, suggesting that calcium influx occurs during CaV-mediated tail currents on the falling phase of the AP in the axon, regardless of temperature (Kawaguchi and Sakaba, 2015;Pressler and Strowbridge, 2019;Rowan et al, 2014;Sabatini and Regehr, 1996).…”

“…While action potential-evoked Ca V -mediated calcium influx has been observed in the AIS of a range of cell classes, the channels that mediate such influx appear to vary from class to class (Bender and Trussell, 2009; Clarkson et al, 2017; Hanemaaijer et al, 2020; Martinello et al, 2015; Yu et al, 2010). To determine the relative contributions of different calcium channel types during AP-evoked calcium influx in mouse prefrontal pyramidal cells, we made whole-cell current-clamp recordings from layer 5 pyramidal neurons in slices prepared from mice aged 20-30 days old.…”

“…To determine whether calcium release from cisternal organelles is recruited at the AIS during AP generation, we began by comparing AP-evoked calcium influx at the AIS before and after ryanodine application, which at high concentrations (>10 µM) blocks calciuminduced calcium release by preventing the opening of ryanodine receptors (Thomas and Williams, 2012). In contrast to somatosensory cortex layer 5b pyramidal neurons, where calcium stores account for ~50% of AP-evoked calcium transients (Hanemaaijer et al, 2020), ryanodine (20 µM) had a more modest effect in prefrontal cortex, reducing AP-evoked calcium transients to 85.4% of baseline ( Fig. 2A-B, IQR = 79.2-89.4%, n = 17, p = 0.008).…”

“…In many of these cells, CaV3 channels appear to be interspersed in the AIS with other CaV classes. This diversity is most apparent in neocortical pyramidal cells, where calcium influx has been reported to be mediated by a range of channel types, including CaV1, members of the CaV2 family, and CaV3 (Clarkson et al, 2017;Hanemaaijer et al, 2020;Yu et al, 2010) Across neuronal compartments, the spatial organization of CaVs shapes function by linking spatially-restricted calcium influx to nearby calcium-sensitive processes. In somatodendritic compartments, coupling of CaVs to calcium-activated potassium channels regulates action potential (AP) dynamics (Bock and Stuart, 2016;Irie and Trussell, 2017), EPSP amplitude and summation (Chen-Engerer et al, 2019;Wang et al, 2014), and calcium influx (Jones and Stuart, 2013).…”

“…In many of these cells, Ca V 3 channels appear to be interspersed in the AIS with other Ca V classes. This diversity is most apparent in neocortical pyramidal cells, where calcium influx has been reported to be mediated by a range of channel types, including Ca V 1, members of the Ca V 2 family, and Ca V 3 (Clarkson et al, 2017; Hanemaaijer et al, 2020; Yu et al, 2010)…”