Cellular Ca2 regulation

The free intracellular Ca2+ represents only a small (~0.001 per cent) fraction of total cellular calcium. Within the cells free Ca2+ is very unevenly distributed between intracellular compartments. Cytosolic free Ca2+ concentration ([Ca2+]j) is very low, being in the range of 50-100 nM. Calcium concentration within the ER is much higher, varying between 0.2 and 1.0 mM, and being therefore similar to extracellular Ca2+ concentration, which lies around 1.5-2.0 mM (Figure 5.10). As a result of these concentration differences an extremely large electrochemical driving force keeps the cytosol under continuous 'Ca2+ pressure', as Ca2+ ions try to diffuse from the high concentration regions to the compartment with low free

These distinct compartments, however, are separated by biological membranes (the plasma membrane and endomembrane separate the cytosol from the extracellular and ER compartments, respectively). Movement of Ca2+ between the compartments therefore requires specific systems, represented by several super-families of transmembrane Ca2+-permeable channels, ATP-driven Ca2+ pumps and electrochemically-driven Ca2+ exchangers. The Ca2+ fluxes resulting from the activity of these systems may either deliver or remove Ca2+ from the cytoplasm (Figure 5.11).

Major routes for plasmalemmal Ca2+ entry are provided by voltage-gated, ligand-gated and nonspecific channels (Figure 5.11), which have distinct activation

Schwann Cell Organelle

Figure 5.10 Cellular calcium gradients. Concentration of free calcium differs considerably between the cytosol and extracellular solution and cytosol and intracellular organelles. The [Ca2+]i in the cytosol is ~20 000 times lower than in the extracellular milieu and ~10 000 times lower than within the lumen of endoplasmic reticulum, which creates huge gradients aimed at the cytoplasm and underlying rapid generation of cytosolic calcium signals. See Figure 5.11 for abbreviations

Figure 5.10 Cellular calcium gradients. Concentration of free calcium differs considerably between the cytosol and extracellular solution and cytosol and intracellular organelles. The [Ca2+]i in the cytosol is ~20 000 times lower than in the extracellular milieu and ~10 000 times lower than within the lumen of endoplasmic reticulum, which creates huge gradients aimed at the cytoplasm and underlying rapid generation of cytosolic calcium signals. See Figure 5.11 for abbreviations mechanisms and differ in their Ca2+ permeability. Plasmalemmal voltage-gated Ca2+ channels are the most selective for Ca2+ ions, whereas other channels allow the passage of Ca2+ and other cations; for example AMPA type glutamate receptors are principally ligand-gated Na+/K+ channels, but also display permeability to Ca2+. Due to a very steep concentration gradient for Ca2+ between the

Calcium Homeostasis Channel

Figure 5.11 Molecular cascades of cellular calcium homeostasis. Calcium homeostasis and the calcium signalling system results from concerted interaction of Ca2+ channels (which include plasmalemmal ion channels, ionotropic receptors and intracellular Ca2+ channels), Ca2+ transporters (Ca2+ pumps and Na+/Ca2+ exchanger, NCX) and cellular Ca2+ buffers. Ca2+ channels provide pathways for Ca2+ entry into the cytosol, whereas Ca2+ transporters accomplish Ca2+ translocation against concentration gradients either back to the extracellular space or into the lumen of the ER. Mitochondria also act as dynamic Ca2+ buffers; mitochondrial Ca2+ accumulation occurs through the Ca2+ uniporter (highly selective Ca2+ channel) down the electro-chemical gradient (intra-mitochondrial potential is -200 mV relative to the cytosol); whereas Ca2+ can be released from mitochondria via NCX (Na+-Ca2+ exchanger), or (especially in pathological conditions) via permeability transition pore (PTP). See the text for further details.

Abbreviations: NCX - Na+ /Ca2+ exchanger; PMCA - Plasmalemmal Calcium ATP-ase; Ca2+-BP -Ca2+ binding proteins; InsP3R - Inositol-1,4,5-trisphosphate Receptor/Inositol-1,4,5-trisphosphate-gated Ca2+ channel; RyR - Ryanodine Receptors/Ca2+-gated Ca2+ channel; SERCA - Sarco(Endo)plasmic Reticulum Calcium ATPase. Intra-ER Ca2+ binding proteins also act as Ca2+ dependent chaperones, which are enzymes controlling protein folding into the tertiary structure extracellular space and the cytosol, opening of even a small number of plasmalemmal Ca2+ channels results in a relatively large Ca2+ influx, which may rapidly change cytosolic free Ca2+. The second important source of cytosolic Ca2+ ions is associated with release from the ER, which serves as an intracellular Ca2+ store; in fact the ER Ca2+ store acts as the main source for cytoplasmic Ca2+ signals in nonexcitable cells. From the ER, Ca2+is delivered to the cytosol via two classes of ligand-gated Ca2+channels residing in the endomembrane, namely ryanodine receptors (RyR) and InsP3 receptors (InsP3R) (Figure 5.12).

RyR are activated by cytosolic Ca2+ ions, and therefore act as an amplifier of Ca2+ signals; these Ca2+ channels are generally known as RyR because they are

Ryr Calcium Release

Figure 5.12 Mechanisms of calcium release from the endoplasmic reticulum:

A. Intracellular Ca2+ release channels are represented by two families of Ca2+-gated Ca2+ channels, or Ryanodine receptors (RyRs), and InsP3-gated Ca2+ channels, or InsP3 receptors.

B. Mechanism of propagating intracellular Ca2+ waves is determined by the Ca2+ sensitivity of both RyRs and InsP3Rs; local increases in [Ca2+]i activate neighbouring channels and produce a propagating wave of excitation of ER-resident Ca2+ release channels

Figure 5.12 Mechanisms of calcium release from the endoplasmic reticulum:

A. Intracellular Ca2+ release channels are represented by two families of Ca2+-gated Ca2+ channels, or Ryanodine receptors (RyRs), and InsP3-gated Ca2+ channels, or InsP3 receptors.

B. Mechanism of propagating intracellular Ca2+ waves is determined by the Ca2+ sensitivity of both RyRs and InsP3Rs; local increases in [Ca2+]i activate neighbouring channels and produce a propagating wave of excitation of ER-resident Ca2+ release channels selectively activated (at low concentrations, < 1 ^M) or inhibited (at 50-100 ^M) by the plant alkaloid ryanodine. The activation of RyR is also regulated by the naturally occurring intracellular second messenger cyclic ADP ribose. There are three types of RyRs, the RyR1 (or 'skeletal muscle' type), the RyR2 (or 'cardiac muscle' type) and the RyR3 (or 'brain' type); although their names are misleading, as they are not only expressed in these tissues; moreover RyR3 expression in the CNS is actually relatively minor. RyR1 can establish direct contacts with plasmalemmal voltage-gated Ca2+ channels, and the opening of the latter upon depolarization will also open RyR1 and trigger depolarization-induced Ca2+ release (which does not require Ca2+ entry and Ca2+ interactions with the ER channel). RyR2 and RyR3 can be activated only by an increase in cytosolic [Ca2+]j, thus producing a calcium-induced Ca2+ release.

InsP3R Ca2+ channels are activated by the intracellular second messenger InsP3, which triggers InsP3-induced Ca2+ release. Importantly, InsP3Rs are also regulated by cytosolic free Ca2+, so that elevation of the latter increases the sensitivity of the receptors to InsP3; high (>1 ^M) [Ca2+]j inhibits type 1 InsP3R, but not type 2 and 3.

Elementary Ca2+ release events associated with opening of a single RyR or InsP3R are respectively known as Ca2+ sparks or puffs; summation of the local events produce a global increase in [Ca2+]j. All in all, release of Ca2+ from several ER channels activates neighbouring RyRs and InsP3Rs, thereby creating a propagating wave of ER excitation; by this means Ca2+ signals are able to travel intracellularly for long distances within polarized cells (Figure 5.12 B).

Importantly, the ER and plasmalemma are functionally linked through a specific class of plasmalemmal channels known as'store-operated Ca2+ channels' (SOCCs; this pathway is also known as a 'capacitative' Ca2+ entry). The latter provide for additional Ca2+ influx in conditions when the ER is depleted from Ca2+; this additional influx helps to replenish the ER Ca2+ stores.

Upon entering the cytoplasm, many Ca2+ ions are immediately bound by Ca2+ -binding proteins (e.g. calbindin 28K), which determine the Ca2+-buffering capacity of the cytoplasm; Ca2+ ions that escape binding, and therefore stay free, generate an intracellular Ca2+ signalling event. The cytoplasmic buffering capacity of different cells varies substantially; for example in a cerebellar Purkinje neurone only 1 out of 4000 Ca2+ ions remain free; in hippocampal neurones this ratio equals 1:70-150.

Excess Ca2+ ions entering the cytoplasm during stimulation are removed by several plasmalemmal and intracellular transporters, which expel Ca2+ from the cytosol against a concentration gradient and prevent the system from overloading. Plasmalemmal Ca2+ extrusion is achieved by Ca2+ pumps (PMCA, plasmalemmal Ca2+ ATPase), which use the energy of ATP hydrolysis to transport Ca2+, and by sodium-calcium exchanger (NCX), which uses the electrochemical gradient of Na+ ions as the driving force for Ca2+ efflux (extrusion of every Ca2+ ion requires entry of 3-4 Na+ ions into the cell, and is dependent on the Na+ concentration gradient maintained by the activity of Na+-K+ pumps). A sizeable amount of Ca2+ is also removed from the cytosol by active uptake into the lumen of the ER via the SERCA pumps (Sarco(Endo)plasmic Reticulum Ca2+ ATPases) residing in the endomembrane. The activity of SERCA pumps is strongly regulated by the free Ca2+ concentration within the ER, and depletion of the ER from Ca2+ ions significantly increases the capacity of the SERCA to pump Ca2+ ions.

All these extrusion systems are assisted by the mitochondria, which are endowed with a very selective Ca2+ channel known as the 'Ca2+ uniporter'. As the mito-chondrial inner membrane is very electronegative compared to the cytosol (up to -200 mV), elevations of cytosolic Ca2+ above ~0.5 ^M drive Ca2+ ions along the electrochemical gradient into the mitochondria. On entering mitochondria, Ca2+ ions activate ATP synthesis and provide a mechanism for coupling cell stimulation with energy production.

Finally, it should be noted that excessive stimulation of Ca2+ influx into the cytosol has a detrimental effect, being the main mechanism of so-called 'Ca2+

excitotoxicity'. The latter occurs during long-lasting excessive stimulation of Ca2+ entry, e.g. by pathological release of glutamate during brain ischaemia, or by failure of Ca2+ extrusion systems, usually through lack of energy. Long-lasting increases in [Ca2+]j in turn stimulate various enzymatic pathways that initiate apoptotic or necrotic cell death.

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