Aequorin Measurements of Cytoplasmic Free Calcium

Abstract

Abstract 1. Introduction The photoproteins aequorin and obelin generate blue-green light upon binding Ca2+ ions. Aequorin has been used over the past twenty years to measure cytoplasmic free Ca2+ (Cat) in single cells by injecting the cell with purified protein and then simply measuring the light emitted from the cell with a photomultiplier. The technique has been used in giant cells [giant barnacle muscle fibre (1), amoeba (2)] down to single mammalian cells [oocytes (3), and cells from heart (4), liver (5), and adrenal gland (6)]. Populations of mammalian cells have been loaded with obelin by liposome fusion, or with aequorin by various reversible permeabilization procedures such as hypo-osmotic shock, ATP-EGTA, scrape-loading, or spin-loading (for review see ref. 7).Photoproteins have several advantages over fluorescent indicators, includ ing the need for much simpler recording instrumentation and freedom from factors such as autofluorescence, intracellular compartmentalization, rapid leakage of the probe from the cell and buffering of calcium by the probe. Relative disadvantages are the need (at present) to microinject single cells, and lack of sufficient signal to make meaningful Cat images in mammalian cells. Recently it has been possible to compare data obtained with aequorin and fura-2 in single hepatocytes (5, 8, 9), when similar cellular Cat responses were found during hormone-stimulation; a gratifying result in view of the complexities of the intracellular environment and possible sources of spurious probe behaviour.