Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-µul drop of aequorin confined to a plastic dialysis tube of 140µzm diameter located axially. Ionized Ca had a mean value of 20 x 10-9 M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasm was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10-9 M as calibrated against CaEGTA/ EGTA buffer mixtures. With a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]i. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca. © 1976, Rockefeller University Press., All rights reserved.
Dipolo, R., Requena, J., Brinley, F. J., Mullins, L. J., Scarpa, A., & Tiffert, T. (1976). Ionized calcium concentrations in squid axons. Journal of General Physiology, 67(4), 433–467. https://doi.org/10.1085/jgp.67.4.433