Coelenterate photoproteins as indicators of cytoplasmic free Ca2+ in small cells

The Ca2+-activated photoproteins aequorin and obelin are capable of detecting rapid changes in free Ca2+ over the range 10nM-100uM. Whilst they have been used to quantify free Ca transients in giant cells for some time, their use in small mammalian cells has been restricted because of the difficulty of incorporating them into live cells without impairment of cell function. We have developed three methods for incorporating photoproteins into small cells (a) reversible cell swelling (b) membrane fusion and (c) intracellular release from pinocytotic vesicles. Formation of the membrane attack complex of complement (C5b6789), via a specific cell surface antibody to activate complement, causes a rapid increase in cytoplasmic Ca2+ detectable within 5-10 s. It provides a specific method for quantifying cytoplasmic photoprotein. As a result new insights into the role of intracellular Ca2+ in cell physiology and pathology have been established.