3.11: Molecular Aspects of Calcium Ion-Regulated Intracellular Processes (Part 1)

So far we have mainly discussed the routes and means by which the concentration of Ca²⁺ ions in the cytoplasm can be transiently increased and brought back to resting levels. But changing the cytoplasmic Ca²⁺ concentration is not enough. In order to influence the cellular machinery, the Ca²⁺ ions must interact with different proteins, intracellular Ca²⁺ receptors if you like. These intracellular Ca²⁺-receptor proteins must have certain properties in order to function.

1. Their Ca²⁺-affinity must be such that their Ca²⁺-binding sites are essentially unoccupied at resting levels of free Ca²⁺ (~10^-7 M) and occupied at levels reached upon stimulus (generally assumed to be 10^-5 to 10^-6 M). This means that the binding constants K_B^Ca2+ should be ~10⁶ M^-1.
2. We should also remember that Ca²⁺ must exert its function in the presence of a number of other ions; in mammalian cells the intracellular concentration of "free" Mg²⁺ ions is around 1 mM, and that of K⁺ ions around 100 to 150 mM. The receptors must therefore have an adequate selectivity for Ca²⁺.
3. In response to Ca²⁺ binding, a Ca²⁺ receptor must undergo some kind of conformation change that either alters its interaction with other molecules or changes its activity if it is an enzyme.
4. Finally, there are kinetic considerations. In many cells a rapid response is essential, and therefore the receptors must be able to interact swiftly—within milliseconds—with incoming Ca²⁺ ions, and the ions must also be able to depart almost as rapidly.

A few proteins have been discovered that qualify as intracellular Ca²⁺ receptors. The best known of these is calmodulin (CaM), which appears to be present in all eukaryotic cells. Most of the cellular responses elicited by Ca²⁺ appear to result from interactions between the Ca²⁺-calmodulin complex and various other target enzymes and proteins.⁷⁵ Another important Ca²⁺-receptor protein is troponin C (TnC), which occurs in muscle cells and is instrumental in mediating muscle contraction.⁷⁶ These two types of proteins are highly homologous, as we shall see, and may be considered members of a superfamily of closely related intracellular Ca²⁺-binding proteins. This superfamily has been given the name "the calmodulin superfamily," and close to 200 distinct family members are presently known.⁷⁷ Not all members of the superfamily may qualify as Ca²⁺ receptors; some like parvalbumins and calbindins (see Section IV.A) appear to have a role in intracellular transport and/or Ca²⁺-buffering. For others, such as the S-100 proteins⁷⁸ found predominantly in brain tissue, and calcimedins,⁷⁹ isolated from smooth muscle, the biological function is still unclear.

One Ca²⁺ receptor with enzymatic activity is protein kinase C. Its activity is markedly increased in the presence of Ca²⁺, and it has a high calcium-binding constant (see Table 3.2) in the presence of diacylglycerol or phorbol esters.⁸⁰

During recent years, groups interested in the role of Ca²⁺ in secretion and in the control of membrane cytoskeleton have identified some intracellular Ca²⁺/phospholipid-binding proteins that appear to be distinct from the calmodulin superfamily; these include lipocortin, endonexin, calelectrin, p36, and calpactin.^81-83 These membrane-binding proteins are collectively called annexins,⁸⁴ and contain repeated domains distinct from EF-hands. The Ca²⁺ sites are very similar to that observed in phospholipase A₂, as shown by the recently determined x-ray structure of annexin V.¹⁷² A condensed overview of the interaction of Ca²⁺ with intracellular proteins is shown in Figure 3.16. We will now go on to discuss the molecular properties of some of the proteins mentioned above, starting with calmodulin.

Figure 3.16 - Condensed overview of the interaction of Ca²⁺ with intracellular proteins.