Plasma Membrane Ca²⁺ Channels
The plasma membrane has a variety of Ca²⁺ entry channels that are characterized by their mechanisms of activation.

Voltage-operated channels (VOCs): A family of channels that open in response to membrane depolarization to mediate the selective entry of Ca2+.5 The multiple types (L, T, N, P/Q and R) are classified on the basis of their kinetics and pharmacological properties: • L-type (long-lasting, activated by high voltage, sensitive to Verapamil and 1,4-dihydropyridines such as Nifedipine. The non-skeletal muscle L channels are blocked by FS-2 and Calciseptine) • N-type (neuronal, transient, activated by high voltage, sensitive to w-conotoxin GVIA) • P/Q-type (long-lasting, low-voltage activated, sensitive to w-agatoxin and funnel web spider toxin, FTX-3.3) • R-type (activated by high voltage) • T-type (transient, low-voltage activated, sensitive to mibefradil) Receptor-operated channels (ROCs): Ca2+ channels that are opened by the binding of specific agonists usually neurotransmitters such as glutamate (the NMDA receptor) or ATP. Store-operated channels (SOCs): Many cells have SOCs in the plasma membrane that are opened by the emptying of the internal stores.6 Just how empty stores communicate with the membrane SOCs is still unclear, current proposals include a calcium influx factor (CIF) or information transfer through a direct protein-protein interaction.7

Intracellular Ca²⁺ Channels
The endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) has two families of intracellular channels responsible for releasing Ca²⁺ from this internal store.^1,8

Inositol 1,4,5-trisphosphate (InsP3) receptors: Agonists acting through G-protein-linked or tyrosine kinase-linked receptors stimulate phospholipase C (PLC) to generate the second messenger InsP3 which then diffuses into the cytoplasm to release stored Ca2+ by binding to InsP3 receptors.1,9 The enzyme PLC can be activated by Pasteurella multocida toxin and inhibited by U-73122 and Neomycin. The InsP3 receptor can be activated by Adenophostin A, Furanophostin and Ribophostin but is inhibited by Heparin, Xestospongin and 2-APB. Ryanodine receptors (RYR): Originally described in muscle cells, these RYRs are also found in neurons and other cell types.9,10 Cyclic ADP ribose is a putative second messenger for regulating the activity of RYRs.11,12 Ryanodine also binds to these receptors and can initiate release by locking the Ca2+ channel in an open configuration. RYR-induced Ca2+ release is activated by Caffeine and 4-Chloro-m-cresol but is inhibited by Dantrolene. The RYR channel is modulated by various associated proteins such as the FK506 binding protein 12 (FKBP12).9 Bastadins can modulate Ca2+ release by interacting with the RYR/FKBP12 complex.13 Sphingolipid Ca2+ release-mediating protein of the ER (SCAMPER): This putative SCAMPER channel is activated by Sphingosine-1-phosphate.

Calcium-Induced Calcium Release (CICR)
The InsP₃Rs and the RYRs are sensitive to Ca²⁺ through both positive and negative feedback effects (see Figure 3). A positive feedback process of Ca²⁺-induced Ca²⁺ release (CICR) is of critical importance in generating and shaping Ca²⁺ signals. Once the channels open a microdomain of Ca²⁺ begins to build up and the channels then close as the positive feedback effect is replaced by a negative feedback response. An important function of InsP₃ and cyclic ADP ribose is to increase the Ca²⁺ sensitivity of the intracellular channels such that the cytoplasm becomes an excitable medium capable of generating Ca²⁺ spikes and waves.

Elementary and Global Aspects of Ca²⁺ Signaling
When cells are stimulated, the global Ca²⁺ signal often appears as repetitive spikes, often organized into regenerative waves which can sometimes take on complex spiral patterns as described in Xenopus oocytes.^2,14 These global responses are built up from the elementary events represented by the opening of either single or localized groups of InsP₃ and RYR channels.¹⁵ Sensitive imaging techniques have begun to reveal these elementary events, e.g. Ca²⁺ sparks in muscle cells and puffs in Xenopus oocytes.^15-17 Regenerative Ca²⁺ spikes and waves are produced by coordinating these elementary events through the process of CICR.

Ca²⁺ OFF Mechanisms
The Ca²⁺ OFF mechanisms depend on pumps that remove the Ca²⁺ signal during the recovery from stimulation.⁴ The surface membrane has a Na⁺/Ca²⁺ exchanger (found mainly in excitable cells) and the ubiquitous plasma membrane Ca²⁺ ATPase (PMCA). The latter is regulated by a variety of factors including calmodulin, acidic phospholipids, and protein kinases A and C (PKA and PKC). The internal stores have a sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) whose activity is inhibited by agents such as thapsigargin, cyclopiazonic acid and BHQ.⁸

The mitochondria also play an important role in Ca²⁺ signaling in that they sequester Ca²⁺ rapidly during the recovery phase and then release it back slowly when the cell is at rest. They are particularly effective when they lie close to the Ca²⁺ release channels.¹⁸ Uptake of Ca²⁺ into the mitochondria is inhibited by Ru360. If the mitochondrion accumulates too much Ca²⁺ it forms a permeability transition pore, which collapses the transmembrane potential leading to the release of cytochrome c and the initiation of apoptosis. The apoptotic regulatory proteins that function as death antagonists (Bcl-2 and Bcl-X_L) or death agonists (Bax, Bak and Bad) may act by modulating the way the mitochondrion handles Ca²⁺.

Ca²⁺-Binding Proteins
Cells have a variety of Ca²⁺-binding proteins functioning either as buffers to shape the cellular response or as sensors to carry out the messenger role of Ca²⁺.

Ca²⁺ Buffers
Both the cytoplasm and the lumen of the ER/SR have proteins capable of buffering Ca²⁺. As Ca²⁺ is pumped into the lumen of the ER/SR, it is buffered by storage proteins such as calsequestrin and calreticulin which have a low affinity (K_d in the mM range) but a high capacity (approximately 50 Ca²⁺ ions bound/molecule). When Ca²⁺ enters the cytosol it is rapidly buffered by proteins such as calbindin, calretinin, and parvalbumin. More than 90% of the Ca²⁺ entering the cell is bound to these buffers with the small remainder representing the stimulus-evoked elevation of Ca²⁺ responsible for activating the Ca²⁺ sensors.¹⁹

Ca²⁺ Sensors
Ca²⁺ sensors such as the EF-hand proteins, annexins²⁰ and the S100 proteins mediate the intracellular effects of Ca²⁺.

The EF hand proteins such as troponin C (TnC) and calmodulin (CaM), which have a characteristic Ca²⁺-binding domain between two helices (named after the E and F a-helices of parvalbumin), are the major Ca²⁺ sensors. TnC functions in skeletal and cardiac muscle whereas CaM has a much more general role. CaM exerts some of its effects by stimulating a multifunctional CaM-dependent protein kinase II (CaMKII) or a CaM-dependent protein phosphatase known as calcineurin.²¹ The function of CAM can be antagonized by W-5, W-7, W-12 and W-13. The immunosupressant drugs FK506 and Cyclosporin A can inhibit Calcineurin.

The annexins represent a heterogeneous family that share a common property of interacting with membranes in a Ca²⁺-dependent manner.²⁰ Since they have a low affinity for Ca²⁺, their action seems to be restricted to domains near membranes where Ca²⁺ channels create localized high elevations of Ca²⁺. Annexins have been implicated in the control of phospholipase A₂, cytoskeletal reorganization, vesicle movement and some may function as Ca²⁺ channels.

Calcium Ionophores
One of the simplest methods of artificially raising the level of intracellular calcium is to use ionophores such as A23187 and Ionomycin.

Caged pounds
A number of calcium signaling Comcomponents are available as caged compounds. They are introduced into cells as inactive precursors and then released by illumination with near UV light. Some of these caged compounds have been produced as membrane-permeant acetoxymethyl (AM) esters. The InsP₃ receptor can be activated using either Caged InsP₃or Caged GPIP₂. Sudden increases in intracellular calcium can be obtained using a variety of caged calcium compounds.

Calcium Probes
Intracellular calcium is measured by introducing a variety of either single-wavelength (e.g. QUIN 2, FLUO 3) or dual-wavelength indicators (FURA 2, INDO 1).

Calcium Buffers
It must be remembered that the calcium probes mentioned above act by binding calcium and thus function as buffers, especially if large amounts are loaded into cells.¹⁹ Both EGTA and BAPTA can be used to increase the normal buffering capacity of the cell.