Measurement of Ca²+ entry using ⁴⁵Ca²+

For decades, the measurement of extracellular Ca(2+) ((40)Ca(o) (2+)) entry into cells, using (45)Ca(2+) as carrier, has been a widely used technique. One of the first studies was performed by Hodgkin and Keynes to measure the rate at which (40)Ca(2+) labeled with (45)Ca(2+) crosses the surface membrane of resting squid axons, or during nervous activity. In a few large excitable cells, it was also possible to measure Ca(2+) entry indirectly through the measurement of inward currents through Ca(2+) channels under voltage-clamp conditions; however, Ca(o) (2+) entry into small excitable cells could only be measured using (45)Ca(2+). With the improvement of patch-clamp techniques, Ca(o) (2+) entry was measured via the analysis of single-channel or whole-cell inward currents through Ca(2+) channels. The first detailed study of Ca(2+) channel currents in small mammalian excitable cells was performed in 1982 in bovine adrenal medullary chromaffin cells, in the laboratory of Erwin Neher, where patch-clamp methodologies were developed. As patch-clamp techniques evolved, it seemed that the measurement of Ca(o) (2+) entry using (45)Ca(2+) would no longer be useful. Isotopes and scintillation fluids are expensive, administrative controls for good laboratory practices are always increasing, and the manipulation of isotopes becomes a growing nuisance. On the other hand, the time resolution of techniques using (45)Ca(2+) entry are in the range of seconds, whereas the patch-clamp measurement of inward Ca(2+) currents is in the range of milliseconds, three orders of magnitude lower.