The Ca 2+ code

One of the modular components of many signal pathways is what deserves to be called the Ca²⁺ code.

“Of the approximately 1,400 grams of calcium that are in the human body, less than 10 grams manage to escape being trapped in the skeleton and teeth. These few grams might be an insignificant quantity, but they are extraordinarily significant qualitatively. They circulate in the blood and extracellular spaces, and penetrate cells to regulate their most important activities” (Carafoli, 2003:

326, my italics).

The versatility of calcium as an intracellular “second messenger” has led some authors to talk about its “universality” as a signal. This ubiquitous intracellular signal is held to be responsible for controlling multiple cellular processes throughout the life of eukaryotic cells from fertilisation to apoptosis, including embryonic pattern

formation, cell differentiation and cell proliferation (Berridge et. al., 2000). “... the Ca²⁺ signal is important in cells from their origin to their death. It controls the creation of cells at fertilisation, masterfully guides them from infancy through adulthood to old age, and finally assists them at the time of their demise” (Carafoli, 2003: 331).

Cells at rest have a low concentration of calcium ions. But when the

concentration rises to specific threshold levels many different functions and cellular responses can be activated.

One of the main questions calcium researchers are asking themselves is: how can these elevations of Ca²⁺ concentration regulate so many processes? Part of the answer lies in the versatility of the Ca²⁺ signalling system in terms of speed, amplitude, and spatio-temporal patterning (Berridge et. al., 2000: 11). But another part of the answer lies in what we have already said, that no single component of a signal-transduction network is by itself the regulator of a cellular response, it is rather one of many mediators.

Actually, it is not the simple linear rise in ion concentration that informs the system and triggers a response. It is rather the fluctuation of complex concentration thresholds.

For this purpose cells employ a sophisticated and extensive repertoire of signalling components, which comprises a “Ca²⁺ signalling toolkit” that can be

assembled in combinations to create signals with widely different spatial and temporal profiles (Berridge et. al., 2000; Carafoli, 2003).

Ca²⁺ signals are generated by using both internal and external sources of Ca²⁺. The internal stores are held within the membrane systems of the endoplasmic reticulum (or the equivalent organelle in muscle cells, the sarcoplasmic reticulum) and within the mitochondrion. The external sources come of course from the

extracellular environment. Release from these internal stores and recruitment from the environment is achieved through various channels that respond to signals (see figure No. 6). There seems to be reciprocal interactions and cooperation between the different organelles and channels in modulating specific patterns of Ca²⁺

concentrations. For example, the endo(sarco)plasmic reticulum provides the Ca²⁺ that enters the mitochondria, which in turn modifies the Ca²⁺ feedback mechanisms that regulate Ca²⁺ from the endo-sarcoplasmic reticulum (Berridge et. al., 2000: 14).

“Environmental” signals indirectly induce some of the channels that let Ca²⁺ in and out of the cytosol, contributing in this way to configuring specific patterns of concentrations of free ions.

Figure No.6. Internal and external Ca²⁺ sources. Taken from Carafoli (2003).

It is important to stress here that when we are talking about a Ca²⁺ signal we are not talking about a Ca²⁺ ion per se but about a “spatio-temporal pattern”. The digital signals represented by single Ca²⁺ ion constitute an analogical sign represented by spatio-temporal patterns of specific threshold concentrations.

Excellent and detailed reviews on the many families and isoforms of components involved in the Ca²⁺ code, and on the subtle complexities of these signalling systems can be found in Berridge et. al. (2000), Weeb and Miller (2003) and Carafoli (2003).

In summary, the Ca²⁺ signalling components include:

1) The sources and stores of Ca²⁺ ions, i.e.: the extracellular space, the endoplasmic reticulum (or sarcoplasmic reticulum), the mitochondria, buffer molecules, the nuclear envelope, and the cytosol, where the signals are configured.

2) Channels, pumps and exchangers (e.g.: Na+/ Ca²⁺), i.e.: membrane-intrinsic proteins that transport Ca²⁺ ions across membranes. The channels are activated or disactivated (directly or indirectly) by extracellular signals (e.g.: neurotransmitters), other second messengers, voltage differences and by Ca²⁺ itself. Channels possess receptor domains being actually receptor-channels. Usually a consensus of different second messengers and other components, plus Ca²⁺ itself, is required for such an activation. There is a continuous fluctuation of Ca²⁺ concentrations created through the many different in-and/or-out-channels that operate at the different sources of Ca²⁺.

3) Ca²⁺ buffers, i.e.: molecules that intercept free Ca²⁺ ions in the cytosol (or in organelles) and maintain them unavailable until they are required as free ions again, constituting an additional mechanism to give specificity to a given needed pattern.

4) Second messengers, i.e.: different Ca²⁺ mobilising messengers (generated when stimuli bind to cell surface receptors) that cooperate in different specific

analogical consensus that activate or inhibit different mechanisms (e.g.: channels) for modulating influx and outflux of Ca²⁺ in the cytosol. The different Ca²⁺ mobilising messengers can coexist in cells where they seem to be controlled by different receptors that respond to specific signals (Berridge et. al., 2000: 12).

5) Sensor or decoding molecules, i.e.: proteins which respond to a given Ca²⁺

concentration pattern. By binding Ca²⁺ ions, sensor-molecules undergo a pronounced conformational change that allows them to continue the cascade towards specific effectors, usually protein kinases which alter other proteins, translating in fact the calcium message into the phosphorylation code and thereby directing the cascade

towards a specific response. A major family of these molecules is the family of EF-hand proteins which include hundreds of members, of which calmodulin is the most thoroughly investigated. There is a group of EF-hand proteins which are collectively called “neuronal Ca²⁺ sensors” which mediate neuronal functions such as the release of neurotransmitters (Carafoli, 2003: 330).