The human body contains an average of 1.4 kg of calcium, which is mainly bound in bones and teeth.[37] Not even 1 % of the calcium within a human is available outside the skeleton in its ionic form but this small amount occurs to be one the most important signalling agents in living systems.[37] Every aspect of life, from its beginning to its death, is regulated by Ca2+.[25,38–40] Already in 1883 S.RINGER discovered the significance of Ca2+ on the contractibility of heart muscles, when no contraction of hearts took place in distilled water but was observed in London tap water.[39,41] Following RINGERs finding, a tremendous variety of Ca2+-regulated processes was detected, including fertilisation[39,40,42], cell growth[43], gene expression[44], hypertrophy[45] and apoptosis[25,38]. Ca2+-dependent regulation of processes within cells demands a carefully adjusted equilibrium between uptake of Ca2+ into the cytosol and its removal out of the cell or storage in the respective organelles like the endoplasmic reticulum (ER) or mitochondria at all times (Figure 2.1).[40,46] The efficiency of calcium signalling is based on the enormous gradient between cytosolic (~10‒7M) and extracellular (~10‒3M) Ca2+
concentration. Activation of a cell via a primary stimulus opens Ca2+ channels, enabling rapid Ca2+ uptake of up to 10-6M within milliseconds.[5,39,47] For precise concentration control of free Ca2+ a multitude of Ca2+-binding proteins has evolved, which trigger cell activation by a primary stimulus. These proteins are divisible in two categories: Ca2+ -buffering and transporting proteins and Ca2+ sensors.[46]
Proteins responsible to transport or buffer Ca2+ are generally located in organelles, integrated into membranes or in the cytosol. The soluble cytosolic proteins can store high quantities of Ca2+ (i.e. parvalbumin, calsequestrin).[5,46,48] Proteins located intrinsically to membranes can form channels, pumps or exchangers to transport Ca2+
through membranes. These Ca2+-transporting proteins regulate the membrane potential and concentration gradients and by modulating the Ca2+ concentration in the cytosol lead to signal transduction.
Ca2+ channels typically transport Ca2+ out of the extracellular space into the cell or enable removal of Ca2+ out of the ER or sarcoplasmic reticulum (SR). Channels positioned in the plasma membrane (PM) are triggered to open by extra- or intracellular binding of ligands or voltage change, while channels in the ER/SR regulate Ca2+-induced Ca2+ release, meaning that Ca2+ regulates itself. Still, this kind of control requires further ligands. Two of the best-known ligands are inositol-(1,4,5)-triphosphate (IP3), that binds to the IP3 receptor (IP3R), and cyclic adenosine diphosphoribose (cADP ribose), which activates the Ca2+-sensitive ryanodine receptors (RyR).[38,46,47]
Synthesis of Ca2+ Sensor-labelled Membrane Components
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Figure 2.1 Schematic illustration of control proteins in eukaryotic cells depicting Ca2+ transport via channels, pumps and exchangers. No differentiation between ligand and voltage gated channels is performed. Picture based on [46].
The second operation mode of membrane-intrinsic proteins is transportation of Ca2+ as ion pumps. A commonly discussed pump in eukaryotic cells is the Ca2+ ATPase.
Within the ER membrane the sarco/endoplasmic reticular Ca2+ ATPases (SERCA pumps) convey Ca2+ uphill against the gradient into the ER, hydrolysing one ATP molecule to transport two calcium ions. Fulfilling the same task, the ATPase in the plasma membrane, the plasma membrane Ca2+ ATPase (PMCA) pump, reduces the cytosolic Ca2+ concentration by pumping Ca2+ out of the cell. In contrast to SERCA pumps the PMCA pumps consume one ATP molecule per Ca2+.[46,49,50]
The last type of Ca2+-binding and transporting proteins are the exchangers. Two examples are the Na+/Ca2+ exchangers in the inner mitochondrial membrane (MNCX) and in the PM (NCX). MNCX removes one Ca2+ out of the mitochondrial matrix in exchange for two Na+, resulting in an electrically neutral process. In contrast, NCX substitutes one Ca2+ by three Na+, making it responsive towards voltage differences and Na+ and Ca2+ transmembrane gradients.
The second category of Ca2+-regulating proteins comprises of the Ca2+ sensors. The best-characterised sensors are the proteins of the EF-hand protein family. For Ca2+-binding the helix-loop-helix motif is used, in which the on average 12 amino acid short loop binds Ca2+ via oxygen-rich side chains like aspartate or glutamate.[46,51]
Synthesis of Ca2+ Sensor-labelled Membrane Components
The most frequently mentioned Ca2+ sensor exhibiting the EF-hand motif is calmodulin (CaM). The structure of this small (~148 amino acids) pervasive protein is highly conserved and has barely changed over the last 1.5 billion years.[38,52,53] CaM is formed like a dumbbell featuring a flexible joint in the middle, connecting two roughly balanced domains with two EF-hands each. Containing this motif, CaM exhibits distinct Ca2+ affinities (Kd = 5 · 10-7M to 5 · 10-6M) with four Ca2+-binding sites, located between the two α-helices of each EF-hand.[53] The Ca2+-free (apo) CaM is arranged in a closed conformation, shielding the hydrophobic residues from the polar surrounding (Figure 2.2, A). Complexation of Ca2+ alters the conformation to an open position, exhibiting hydrophobic areas of each domain (Figure 2.2, B).[53] The exposed methionine-rich hydrophobic surfaces are now enabled to bind target enzymes by wrapping around their amphipathic domains, i.e. myosin-light-chain kinase (MLCK, Figure 2.2, C).[38]
Ca2+-bound CaM (Ca2+4-CaM) plays a crucial role in activation of a variety of enzymes.
One example is the phosphorylase kinase, which is dependent on Ca2+4-CaM to trigger cleavage of glycogen under glucose release.[54] Furthermore, MLCK is activated by Ca2+4-CaM, leading to phosphorylation of the myosin light chain, hence enabling smooth muscle contraction.[55] As a conclusive example the Ca2+4-CaM-dependent protein kinase II (CaM kinase II) is to be mentioned. This kinase occurs in neurons in high concentrations and is responsible for activating further enzymes like calcineurin (CaN), tyrosine hydroxylase (TYH) and nitric oxide synthases (NOOSs).[56]
Figure 2.2 CaM in its closed form, with shielded hydrophobic domains (A); Ca2+4‒CaM in the open form (B); Ca2+4‒CaM complexed a MLCK (red helix) with its hydrophobic binding domain (C). Pictures taken from [57].
Synthesis of Ca2+ Sensor-labelled Membrane Components
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