TRPA1-dependent calcium transients and CGRP release in DRG neurons require extracellular calcium

Abstract

Shang et al. (2016. J. Cell Biol.https://doi.org/10.1083/jcb.201603081) reported that activation of lysosomal TRPA1 channels led to intracellular calcium transients and CGRP release from DRG neurons. We argue that both findings are more likely due to influx of insufficiently buffered extracellular calcium rather than lysosomal release.

Reply to “TRPA1-dependent calcium transients and CGRP release in DRG neurons require extracellular calcium”

“…Indeed, no inward Ca 2+ currents or [Ca 2+ ] i rise through plasma membrane Ca 2+ permeable channels, such as voltagegated Ca 2+ channels, could be detected in the Ca 2+ -free bath that contained 1 mM EGTA (Zhang and Zhou, 2002). Gebhardt et al (2020) also assumed that there would be some "loosely attached calcium ions" that bind to "the anionic extracellular moieties of membrane proteins" and that they would become free in the 1 mM EGTA solution and contribute to Ca 2+ entry through the plasma membrane TRPA1 channels. We find this assumption to be without any evidence to support it.…”

“…Here, we would like to address their concerns regarding the lysosomal TRPA1-mediated intracellular calcium transients in dorsal root ganglion neurons. Gebhardt et al (2020) first questioned our conclusion that TRPA1 mediates both Ca 2+ influx through plasma membrane and Ca 2+ release from lysosome-related internal Ca 2+ store in dorsal root ganglion (DRG) neurons (Shang et al, 2016). The reason for raising this doubt is that although they were able to reproduce our result that the TRPA1 agonist allyl isothiocyanate (AITC) triggers a rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ) of DRG neurons bathed in the Ca 2+ -free extracellular solution containing 1 mM EGTA, this effect was largely abolished with the solution that contained 10 mM EGTA.…”

“…The reason for raising this doubt is that although they were able to reproduce our result that the TRPA1 agonist allyl isothiocyanate (AITC) triggers a rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ) of DRG neurons bathed in the Ca 2+ -free extracellular solution containing 1 mM EGTA, this effect was largely abolished with the solution that contained 10 mM EGTA. Based on this observation, Gebhardt et al (2020) concluded that 1 mM EGTA must not be enough to buffer the residual Ca 2+ present in the nominally Ca 2+ -free physiological solution made using commercial American Chemical Society grade constituents. Gebhardt et al (2020) are correct in citing the reference from Patton et al (2004) that the Ca 2+ concentration in nominally Ca 2+ -free solutions can be as high as 12 µM, a fact that the majority of researchers in the Ca 2+ field have been fully aware of for decades.…”

“…Based on this observation, Gebhardt et al (2020) concluded that 1 mM EGTA must not be enough to buffer the residual Ca 2+ present in the nominally Ca 2+ -free physiological solution made using commercial American Chemical Society grade constituents. Gebhardt et al (2020) are correct in citing the reference from Patton et al (2004) that the Ca 2+ concentration in nominally Ca 2+ -free solutions can be as high as 12 µM, a fact that the majority of researchers in the Ca 2+ field have been fully aware of for decades. This is why Ca 2+ chelators, such as EGTA, HEDTA, or BAPTA, are often included in the Ca 2+ -free solutions.…”

“…These multiple pieces of hard evidence indicate that not only does TRPA1 activation, in addition to Ca 2+ influx, mobilize Ca 2+ from lysosomerelated stores, but also that the internally generated Ca 2+ signals are able to support several known physiological functions of this channel (Shang et al, 2016). Lastly, Gebhardt et al (2020) questioned the GPN effect. They assumed TRPA1 to be permeable to protons by citing Ye et al (2018).…”