Measurement of Ca2+ Entry Using 45Ca2+

For decades, the measurement of extracellular Ca²⁺ (⁴⁰ Ca_o ²⁺ ) entry into cells, using ⁴⁵ Ca²⁺ as carrier, has been a widely used technique. One of the first studies was performed by Hodgkin and Keynes (1 ) to measure the rate at which ⁴⁰ Ca²⁺ labeled with ⁴⁵ Ca²⁺ crosses the surface membrane of resting squid axons, or during nervous activity. In a few large excitable cells, it was also possible to measure Ca²⁺ entry indirectly through the measurement of inward currents through Ca²⁺ channels under voltage-clamp conditions (2 ); however, Ca_o ²⁺ entry into small excitable cells could only be measured using ⁴⁵ Ca²⁺ . With the improvement of patch-clamp techniques (3 ), Ca_o ²⁺ entry was measured via the analysis of single-channel or whole-cell inward currents through Ca²⁺ channels. The first detailed study of Ca²⁺ channel currents in small mammalian excitable cells was performed in 1982 (4 ) 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 Ca²⁺ 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 Ca²⁺ entry are in the range of seconds, whereas the patch-clamp measurement of inward Ca²⁺ currents is in the range of milliseconds, three orders of magnitude lower.