Use of ionophore A23187 to measure cytoplasmic Ca buffering and activation of the Ca pump by internal Ca

Abstract

DURING the formation of the mature mammalian erythrocyte, the reticulocyte sheds its nucleus, endoplasmic reticulum and mitochondria leaving a featureless cytoplasm with a high haemoglobin content surrounded by a plasma membrane. All Ca-accumulating organelles are lost and the mature red cell is maintained virtually Ca-free throughout its life¹ by the low Ca permeability of its membrane² perhaps aided by a powerful Ca-extrusion pump³. Most of our knowledge of Ca transport in red cells comes from studies with resealed ghosts, but in this preparation the results are often affected by alterations to the membrane and metabolism of the cell^2–4. The main difficulty of using intact fresh cells in physiological conditions has been the impossibility of increasing their Ca content in a controlled way and of assessing the fraction of Ca which is ionised. Using a divalent-cation ionophore, A23187 (ref. 5), however, we have overcome these difficulties and found that cytoplasmic Ca buffering occurs as if the cell had a single large-capacity, low-affinity Ca buffer and that there are two Ca-translocating sites of equal affinity at the internal surface of the Ca pump.