Physiological [Ca2|[plus]|]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator

doi:doi:10.1038/298478a0

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Nature 298, 478 - 481 (29 July 1982); doi:10.1038/298478a0

Physiological [Ca²⁺]_i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator

Virgilio L. Lew^*, Roger Y. Tsien^*, Cristina Miner^* & Robert M. Bookchin^†

^*Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, UK
^*Present address: Department of Physiology-Anatomy, University of California, Berkeley, California 94720, USA.
^†Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA

The physiological actions of Ca²⁺ as a trigger and second messenger depend on the maintenance of large inward resting Ca²⁺ gradients across the cell plasma membrane. An ATP-fuelled Ca-pump, originally discovered and still best characterized in human red cells¹⁻⁴, is now believed to mediate resting Ca²⁺ extrusion in most animal cells. However, even in red cells, the truly physiological pump-leak turnover rate and cytoplasmic free Ca²⁺ level are unknown. Previous estimates^5,6 were only very imprecise upper limits because normal intact red cells have a minute total pool of exchangeable Ca of less than 1 µmol l^-1 cells⁷; Ca fluxes could not be measured without artificially increasing that pool with ionophores⁸⁻¹⁰ or disrupting the membrane to incorporate Ca buffers⁵. Both procedures leave the membrane considerably leakier than in intact cells. Here, we have increased the exchangeable Ca pool by non-disruptively loading a Ca-chelator into intact cells, using intracellular hydrolysis of a membrane-permeant ester^11,12. The trapped chelator made the free cytoplasmic calcium concentration, [Ca²⁺]_i, an easily defined function of directly measurable total cell Ca. We were then able to establish the physiological steady-state [Ca²⁺]_i and pump-leak turnover rate of fresh cells suspended in their own plasma. If [Ca²⁺]_i was lowered below the normal resting level, the Ca pump rate decreased according to the square of [Ca²⁺]_i, and the inward Ca leak increased. The increase in leak did not develop if the cells were depleted of ATP and ADP.