The [Ca2+]i Concept: from whole Cell to Microdomains
The discovery that Ca ions play key roles in biology, not only as cations that contribute to the generation of currents and to the control of potential at the plasma membrane, but also in the regulation of intracellular functions and activities, stems from the pioneer experiments of Ringer in the late Century and has been uninterruptedly reinforced and expanded even since (see Pietrobon et al., 1990). The mechanisms of the second messenger function of Ca2+, however, have begun to be unravelled in the early seventies. Only at that time, in fact, the basic distinction was made between the total calcium content of the cells, which is high (mmoles/liter, in the same range as the concentration of the cation in physiological extracellular fluids), and the concentration of the free cation in the cytosol ([Ca2+]i) which, because of its equilibrium with physiological activity regulators (proteins, enzymes etc.), is considered as the main actor in cellular control (Pietrobon et al., 1990; Carafoli, 1987; Pozzan et al., 1994). The first measurements of [Ca2+]i, carried out by microimpalement of large cells with Ca2+ specific electrodes, revealed unexpectedly low [Ca2+]i levels, i.e. four orders of magnitude lower than the total cell calcium content. From those measurements the idea first emerged that cells contain molecules and structures capable of binding and segregating Ca2+. The level of what remains free in the cytosol, i.e. [Ca2+]i, is maintained in a relatively narrow range as the result of complex and dynamic equilibria (Pietrobon et al., 1990; Carafoli, 1987; Pozzan et al., 1994). In the following years these seminal observations and ideas have been confirmed by an interrupted stream of findings. Here we would like to emphasize only two of them: the development and introduction into biological research of the fluorescent Ca2+ dyes that, beginning in 1982, have revolutioned the field since they made possible [Ca2+]i measurements in all cells, no matter of their size and origin; and the identification in the cytosol of Ca2+ binding proteins, with their classification into two main families: the E—F hand-containing proteins (over 260 proteins nowadays!) and the annexins (Huzinken, 1994). Since both these families of proteins bind Ca2+ with high affinity (Kds in the 10−7 − 10−6 range, i.e. the [Ca2+]i range revealed experimentally by the dyes in intact cells), the opinion was tacitly shared that all the Ca2+-controlled events of the cytosol had to be of high affinity. As usual, diverging opinions about the existence in the cytosolic compartment of low affinity Ca2+ controlled phenomena were expressed also at that time. They however failed to have much impact as long as they were supported more by hypotheses than by direct experimental evidence. In addition, it was difficult to envisage the existence within the cell of cytosolic microdomains containing high [Ca2+] while the average [Ca2+]i, revealed by the dyes, was measured in the usual 10−7 – 10−6 M range. After all, the diffusion of Ca2+ in a solution is fast (∼350 Μm2/sec) and no mechanisms could therefore be envisaged that could restrain the rapid dissipation of high [Ca2+]i domains, if formed.
KeywordsSynaptic Vesicle Active Zone Purkinje Neuron Direct Experimental Evidence Presynaptic Membrane
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