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

Abstract

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 cells^1–4, 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 ionophores^8–10 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.