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Ca2+ Chemistry, Storage and Transport in Biologic Systems: An Overview

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Calcium ions play a critical role in most if not all cellular processes. It has even been demonstrated that Ca2+ currents in root tips, in combination with gravity, are responsible for their downward growth.1 Most of these effects are mediated by both temporally and spatially tightly controlled changes in cytosolic free Ca2+ brought about by activation of Ca2+ influx pathways in the cell membrane or by activation of intracellular Ca2+ release channels, and countered by transporters acting as Ca2+ pumps. Voltage-gated Ca2+ channels are a preeminent class of plasma membrane proteins providing regulated Ca2+ influx, and the remainder of the volume is dedicated to their many different facets. In this introductory chapter, we will briefly review other aspects of cellular Ca2+ homeostasis. We will review some of the chemical properties of Ca2+ that are important to its role in biology, we will review intracellular Ca2+ stores, and we will review other Ca2+ handling proteins. Due to space limitations we will not be able to refer to the original literature in most cases, but have to limit ourselves to a relatively small number of recent reviews that could provide the reader further guidance into the many exciting and sometimes controversial topics of current research in Ca2+ transport and storage in cells.

Why Ca2+: Unique Chemical and Physical Features

The chemical features and physical characteristics of the doubly charged Ca2+ contribute favorably to its role in biology. This has been extensively reviewed by Williams2,3 and a brief synopsis is provided here.

Ca2+ displays a highly ionic character in its bonding. The covalent contribution in bond formation increases as one moves across the series from Ca2+ to Zn2+. The highly ionic character of Ca2+ in bond formation restricts its interactions to liganding oxygen donating groups such as carbonyl, carboxyl, alcohols and ethers. Divalent cations that display highly covalent bond formations, such as Cd2+, interact with stronger donating groups such as nitrogen and sulfur donors, and are thus removed from competing with Ca2+ for binding.

Ca2+ is a rather large ion with a crystal ionic radius of 0.99 Å. This is larger than the majority of divalent cations including those in the third row of the periodic table from Mn2+(radius of 0.80Å) across to Zn2+ (radius of 0.74Å). Cd2+ is very close in size with a radius of 0.97Å. Relative to the other ions in the second period, Ca2+ is larger than Mg2+ (radius of 0.66Å), however, smaller than Sr2+ (01.12Å) and Ba2+ (1.34Å).

In terms of the binding capability, ionic radius is an important determinant as to the strength and type of ligand that will bind to a particular ion. If this alone was the determining factor for the binding of potential ligands, then it would follow that smaller divalent cations would exhibit the strongest ligand binding due to a stronger electrostatic field. This, however, is not the case. Smaller ions necessitate closer association of donor groups in three-dimensional space. This restricts the binding of ligands to smaller ions due to steric hindrance within the liganding molecule. However, if a divalent cation is too large, the reduction in electrostatic field causes a decrease in binding. Ca2+ provides the best balance between the two opposing forces; steric hindrance and electrostatic field intensity.

Ionic radius also determines the solubility of a particular ion with other ions. Mg2+ can pack tightly into the small holes created in the lattice of smaller anions such as hydroxide and fluoride ions. Ca2+, however, is too large to fit into the holes in the lattice structure created by OHand F- ions. Consequently, Mg2+ rather than Ca2+, tends to precipitate out with small anions. Ca2+ precipitates out with larger anions such as phosphates and carbonates or polyanions such as nucleic acids and acidic proteins.

Ca2+ Evolution: A Rejection from Cytoplasm

An interesting theory on the evolution of the role of Ca2+ in biology has been presented by Williams,2,3 based on the ability for Ca2+ to precipitate organic anions. From life's very beginnings, it was essential that Ca2+ be separated from organic anions to avoid precipitation. Thus, Ca2+ had to be rejected from the cytoplasm of the earliest cells. This initial simple rejection of Ca2+ is still observed for all organisms, ranging from prokaryotes to multicellular eukaryotes, where intracellular free Ca2+ concentrations are maintained on the order of 10-7M. In the earliest forms of life, namely prokaryotes, Ca2+ was simply rejected to intracellular levels on the order of 10-5M. Little function for intracellular Ca2+ is found in anaerobic bacteria to this day.

With the development of eukaryotes came the compartmentalization of the cytoplasm. It was then necessary for eukaryotic cells to develop intracellular signaling in order to coordinate function and placement of these organelles. The Ca2+ features outlined above makes Ca2+ an excellent second messenger, fast kinetics, and easy liganding ability. Therefore, eukaryotic cells started to use Ca2+, and the previously set up Ca2+ electrochemical gradient from prokaryotes, for coordination of events within the cell. It is not until the development of unicellular eukaryotic cells, such as yeast, that we observe intracellular Ca2+ binding proteins. Emergence for the use of Ca2+ precipitates as external support and protective structures begin in eukaryotes with calcium carbonate shells. Multicellular eukaryotes then develop organized internal calcium phosphate precipitates in the form of bone, which act not only to provide structural support, but act as a Ca2+ reservoir for maintenance of circulating blood Ca2+ levels. The progression of the extensive use of Ca2+ is then based on its initial rejection from prokaryotic cells, creating a gradient for Ca2+ entry. This entry, in combination with the unique binding characteristics of Ca2+, allowed eukaryotic cells to use this divalent cation as an essential element for the many processes in which it is used today.

Ca2+ Signaling and Storage: The Endoplasmic Recticulum

The endoplasmic recticulum is generally the predominant intracellular Ca2+ store. The ER is critical not only for Ca2+ storage, but release of Ca2+ from the ER is responsible for rapid transmission of Ca2+ signals from the periphery to the center of the cell and for local Ca2+ signaling.4 Interestingly, changes in ER Ca2+ handling is thought to be involved in neural ageing5 and improper ER Ca2+ control has pathogenic implications.6 Resting ER free Ca2+ concentration is thought to be on the order of several hundred μM,7 in addition, lumenal ER Ca2+ is stored by high capacity-low affinity Ca2+ buffering proteins including calsequestrin and calreticulin.8

This resting level of lumenal ER free Ca2+ is the result of a balance between Ca2+ uptake via sarcoplasmic-endoplasmic type ATPases (SERCAs)9 and release. Ca2+ is released from the ER in either a stimulated or passive manner.

Stimulated release occurs through the activation of two receptor types; ryanodine receptors(RyR) and inositol 1,4,5-triphosphate(IP3) receptors discussed in more detail later.8 In addition, other receptors may exist that release Ca2+ via cyclic-ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). Suprastimulation with high levels of Ca2+ releasing agonists reduce ER free Ca2+ to only the tens of μM range.8 Inactivation of Ca2+ release channels by high cytosolic Ca2+ may contribute to this incomplete emptying. Interestingly, sustained submaximal levels of cytosolic IP3 causes only partial transient release of calcium from the ER. Whereas additional levels of IP3 induce more pulses of Ca2+ release. This is known as the quantal Ca2+ release phenonmenon.11 The exact mechanism of this phenomenon, however, remains elusive.

Passive release is less well-defined and has been shown in many cell types to be remarkably dynamic as inhibition of the SERCA pump by thapsigargin often results in a rise in cytosolic free Ca2+ within a few seconds. This passive leak has been shown to be insensitive to inhibitors of both IP3 and RyRs, but responsive to ATP levels.10 Recent evidence has implicated a protein involved in apoptosis, Bcl2, as having a possible role in passive Ca2+ leak from the ER.8

The Role of Ca2+ within the ER

Ca2+ is fundamental for proper function of the ER. Indeed, evidence has accumulated that indicates that even ER structure and integrity are dependent upon lumenal free Ca2+. Reports have indicated that loss of ER lumenal Ca2+ results in ER fragmentation.12 Additionally, it has been shown that high levels of cytoplasmic Ca2+ can result in the loss of continuity of the ER membrane.13 Prevention of ER fragmentation has been observed upon protein kinase C(PKC) activation, which could possibly provide the ER with a protective mechanism upon store depletion with ip3/DAG producing agonists.13,14 However, another group has since then observed that the protective effects of PKC activation occur only when fragmentation is caused by an increase in cytoplasmic Ca2+ and not when caused by ER Ca2+ depletion.15

In addition to Ca2+ handling, the ER is the site of protein processing, storage and transport. Interestingly, many Ca2+ binding proteins found within the ER also act as chaperone proteins. The two roles served by these proteins, chaperone and Ca2+ buffer, are not separate, but are intimately associated. Maturation and proper folding of proteins is affected by depletion of ER Ca2+.8 The formation of chaperone complexes in the ER depends on Ca2+ concentration.16 Additionally, depletion of ER Ca2+ increases protein degradation.17

The ER alone is not responsible for correct trafficking and maturation of proteins. The ER is in constant communication with the Golgi network with proteins and vesicles being transported between the two. Ca2+ has been found to play an important role in this communication. Transport between the ER and Golgi has been shown to require certain levels of cytosolic free Ca2+ and may be dependent upon a local Ca2+ gradient in the cytoplasm separating these two organelles. In addition, transport of vesicles between the ER and Golgi depends on the presence of internal Ca2+, within both the ER and Golgi, as transport did not occur following ER and Golgi emptying with Ca2+ ionophores and the SERCA pump inhibitor thapsigargin.8

Ca2+ also acts to control Ca2+ management within the ER as a feedback mechanism. There are several examples of this phenomenon, however, the most extensively studied is capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCs). It has been found that depletion of lumenal ER free Ca2+ can stimulate the entry of extracellular Ca2+ into the cell in order to refill the ER(reviewed in ref. 18). CCE is discussed in later text. Passive Ca2+ leak out of the ER is also dependent on ER lumenal Ca2+. Observation have indicated that low levels of ER Ca2+ (100 μM) greatly reduced this passive leak, additionally, ER Ca2+ affects ip3R, SERCA activity and even expression of the ER Ca2+ binding protein calreticulin.8

ER Heterogeneity: Ca2+ Tunneling Versus Ca2+ Compartmentalization

Petersen provides an excellent discussion on the topic of ER calcium distribution.11 An outline of this discussion is provided here. Classically, the endoplasmic recticulum has been thought of as one continuous membrane bound enclosure with equal protein expression and equilibrated ion concentrations throughout. ER protein expression, however, is not uniform but heterogeneous with specific localization of proteins into domains. Different SERCA subtypes and Ca2+ release channels are found in different parts of the ER. This differential ER protein distribution is separate from, but related to the topic of ER calcium distribution. Recently, this topic has been the subject of debate. There have been many reports that IP3 induced and Ry/caffeine induced Ca2+ release occur through separate calcium stores. Imaging experiments have indicated that IP3 induced Ca2+ release is spatially distinct, and can be independently emptied, from release induced by Ry/caffeine.4 Currently, however, the classical view of an equilibrated ER Ca2+ distribution is accepted based on the results of diffusion using uncaged Ca2+ within the ER.

Ca2+ Signaling and Storage: Mitochondria

Since the 1960s, it has been known that mitochondria are capable of accumulating Ca2+. The importance of this uptake, however, was thought to be minimal as physiologic increases in cytosolic Ca2+ was seen as being insufficient to activate mitochondrial Ca2+ uptake mechanisms. Interest in mitochondrial Ca2+ uptake was then sparked by experiments using the Ca2+-indicating protein aequorin, targeted to the mitochondrial matrix. These experiments indicated that mitochondrial matrix Ca2+ levels transiently increased following treatment with IP3 liberating agonists.19 Mitochondria exhibit two mechanisms of Ca2+ uptake. Primarily, Ca2+ uptake occurs via a Ca2+ uniporter. The molecular characterization of this uniporter, however, remains elusive. Evidence has indicated that this uniporter functions like a channel with an increase in open probability associated with increased local Ca2+ concentrations(reviewed in 20). Ca2+ uptake through this uniporter is known as the Ruthenium Red (RuR)-sensitive pathway. The driving force for Ca2+ entry through the uniporter is provided by the mitochondrial membrane potential, reported to be -150 to -200mV with respect to the cytosol.21 The uniporter has a rather high Ca2+ dissociation constant (Kd>25 μM),19 much greater than physiologic changes in net cytosolic Ca2+. Mitochondria, however, are subject to much higher cytoplasmic concentrations of Ca2+ in the form of microdomains that are released upon IP3 receptor opening in the ER. In fact, the ER and mitochondria have been found to be very closely associated in space(<100nm), and in Ca2+ communication(see next section).22 A second form of mitochondrial Ca2+ uptake has also been reported and is known as the rapid uptake pathway.23 Little is known about this mechanism of uptake. Mitochondrial Ca2+ uptake is balanced by Ca2+ extrusion through the mitochondrial Na+Ca2+ exchanger, which exhibits different characteristics than the plasma membrane exchanger.21

Mitochondrial Ca2+: Uptake Function and Effects

The main role of mitochondria has long been known to be oxidative phosphorylation.24 Not surprisingly then, mitochondrial Ca2+ levels influence this process. The citric acid cycle is rate limited by levels of dehydrogenases. Expression of these enzymes is upregulated, with associated increase in ATP production, in response to accumulation of mitochondrial Ca2+.25

A more unexpected role of Ca2+ uptake by mitochondria involves the fine tuning and shaping of intracellular Ca2+ signaling. A conventional view of Ca2+ signaling within a cell consists of a wave of Ca2+ propagating across the cytoplasm of the cell. This propagation is thought to occur by CICR through IP3 receptors in the ER. The wave starts with a local increase in cytoplasmic Ca2+. This sensitizes IP3 receptors and enables their activation at lower levels of IP3. The ip3 receptor activates and releases Ca2+, which acts to sensitize the IP3 receptors on adjacent ER membrane. In this manner the wave of Ca2+ is released into the cytoplasm across the cell. The action of mitochondria in shaping of these signals is to restrict the rate of wave propagation and dampen its amplitude as it moves across the cell. Inhibition of mitochondrial Ca2+ uptake results in a constant rate of wave propagation and an increase in the rate of rise and amplitude of the signal.22 In pancreatic acinar cells, mitochondria found along the center line separating the apical and basal portions, has been shown to restrict the spread of Ca2+ waves from one end to the other.26

Under certain conditions, (oxidative stress, adenine nucleotide depletion and increased inorganic phosphate levels) an increase in intra mitochondrial Ca2+ concentration will trigger the formation of a pore complex known as the permeability transition pore (mPTP). This complex is made up by several proteins located at the close contact points between the inner and outer mitochondrial membranes. The function of the mPTP, however, remains to be evaluated. Several theories have been put forward including Ca2+ signal modulation, apoptosis initiation, or Ca2+ release.22

Ca2+ Buffering: Cytosolic and Lumenal

Protein bound calcium accounts for between 95% and 99% of the total Ca2+ load of a cell.27 The proteins that are involved in this binding are collectively known as Ca2+ buffering proteins and belong to the EF-hand family of Ca2+ binding proteins.28 Ca2+ buffering proteins serve several functions within cells. Besides their obvious function to buffer cytoplasmic/lumenal free Ca2+ levels, they can act as Ca2+ delivery proteins, signaling proteins, and chaperones. A soluble cytosolic protein that demonstrates two of these functions is parvalbumin(PV). This high affinity Ca2+ binding protein is found in high concentration in the sarcoplasm of vertebrate fast contracting muscles, where it is involved in muscle relaxation. The process of muscle relaxation occurs when Ca2+ dissociates from troponin C, moves towards, and is transported into the sarcoplasmic recticulum (SR). The sarcoplasm/cytoplasm of cells is not a free space in which Ca2+ can diffuse, but is instead filled with negatively charged proteins capable of binding to Ca2+. Therefore, Ca2+ diffusion occurs rather slowly in these areas. In order to facilitate Ca2+ translocation, Ca2+ is taken up by PV, which moves freely to other locations in the cell. It is generally thought that after muscle contraction, Ca2+ is taken up by PV, which then relocates to deliver the Ca2+ to the SERCA pump of the SR. There has also been evidence for a direct interaction between PV and the SR through the binding of PV to an SR protein. This binding occurs in a Ca2+-dependent manner.29

Ca2+ buffering in intracellular organelles involves high binding capacity, low affinity (Kd.1mM) Ca2+ binding proteins including; calnexin, calreticulin, calsequestrin and endoplasmin.8 ER Ca2+ buffering in muscle cells is primarily a function of calsequestrin.30 The ER of other cell types employs calreticulin, which acts not only in Ca2+ buffering, but as a molecular chaperone protein.31 Lumenal ER Ca2+ binding capacity has been reported to be quite low compared to that of the cytosol. In mouse pancreatic acinar cells, cytosolic Ca2+ binding capacity was observed to be two orders of magnitude greater than that of the ER.32 This high cytosolic binding capacity has been reported for some cell types, while found to be lower in others.11 A complete picture of ER lumenal binding capacity and mobilities of the Ca2+ buffering proteins involved, in both the ER and the cytosol, needs further investigation.

Channels that Lead to an Increase in Cytosolic Ca2+

Temporal and spatial control of increases in cytosolic free Ca2+ is of paramount importance for proper functioning of most cells, and aberrant Ca2+ homeostasis can rapidly result in cell death. Therefore, it is not that surprising that members of several different gene families of Ca2+ permeable channels may contribute to Ca2+ influx into the cytosol. The first members of most of these gene families were discovered in the eighties and nineties, quickly followed by cloning of related gene products; by now with the human and mouse genomic sequencing projects nearing completion, a complete membership list is probably available for most gene families. Many of these channels contributing to Ca2+ influx into the cytosol can be found coexpressed in most cell types, especially in cells of excitable tissues. To reflect the diversity of specific cellular functions, most of the above gene families contain many different but related members, and this complexity is further increased by the presence of different splice variants. Although the occurrence of different splice variants is common, only in very few cases has a physiological significance been established with certainty.

Intracellular Ca2+ Release Channels

Two families of intracellular Ca2+ release channels have been found to date, the IP3 receptor33 and the Ryanodine receptor (RyR),34 each family consisting of three distinct mammalian genes. The IP3 and Ryanodine receptors are among the largest ion channel proteins and form functional tetramers with a MW of about 1.2 and 2 million daltons, respectively.

Ryanodine Receptors34

Three isoforms of mammalian RyR have been cloned: RyR1 is expressed in skeletal muscle (5040 residues), RyR2 is expressed in cardiac muscle (4945 residues), and RyR3 is expressed in skeletal muscle (4871 residues) as well, but in lower amounts than RyR1. RyR1 and RyR2 are critical to proper functioning of the host tissue as knocking out these genes in mice is lethal; in contrast, the RyR3 knockout is relatively normal. The ryanodine receptor functions as an intracellular Ca2+ release channel responding to a depolarization of the plasmalemma, although the mechanism is quite different for the RyR1 and RyR2 isoforms, respectively. In skeletal muscle, membrane depolarization is directly mediated via protein-protein interactions involving the plasmalemma dihydropyridine receptor (i.e., voltage-gated calcium channel) and the RyR1 located in the sarcoplasmic reticulum. In heart muscle, membrane depolarization results in Ca2+ influx via voltage-gated calcium channels that is used as a trigger for further Ca2+ influx by opening RyR2 in the sarcoplasmic reticulum. The conductance properties of RyR channels are regulated by cytosolic Ca2+, Mg2+, ATP and phosphorylation. Channel opening is promoted by low cytosolic Ca2+ (1-10 μM) and inhibited by high Ca2+ (>1 mM); channel opening also appears regulated by luminal Ca2+, but this is less clearly defined. RyR1 is more sensitive to regulation by Mg2+ and free ATP compared with the other two isoforms: free Mg2+ is a potent inhibitor, while ATP activates RyR. In addition to cytosolic Ca2+, Ca2+ release from RyR can also be stimulated by cyclic-ADP-ribose, by caffeine, and by high concentrations of ryanodine. Ryanodine receptors have also been shown to associate with several other intracellular proteins including calmodulin, calsequestrin (the major soluble Ca2+ binding protein in the SR), and immunophilin FK-506 binding proteins. Of all of the effectors of RyR function, Ca2+ is clearly the most significant.

IP3 Receptors33

Three isoforms of mammalian IP3 receptors have been cloned containing around 2700 amino acids each. Isoforms show about 72% homology amongst each other, and, similar to the ryanodine receptor, functional channels are thought to be tetramers of identical subunits. For each subunit, three main domains can be distinguished: the N-terminal ligand binding domain (≈500 residues), followed by a long regulatory domain (≈1600 residues) containing among other things two consensus ATP-binding sites and the binding domains for calmodulin and FK-506 binding protein, and the C-terminal ion channel domain (≈450 residues) that anchors the IP3 receptor to the membrane. The C-terminal ion channel domain shows homology to the ion channel domain of the ryanodine receptor. IP3 opens channels in a cooperative fashion suggesting that each subunit requires IP3 to bind for channels to open; half of the channels are opened at <1 μM IP3. As with ryanodine receptors, cytosolic Ca2+ exerts biphasic effects with potentiation of IP3 effects at low Ca2+ concentration and inhibition of channel activity at high Ca2+ concentration. IP3 receptors are also regulated by ATP in a biphasic manner with low ATP concentrations increasing channel activity and high ATP levels leading to a decrease in channel activity. IP3 receptors show tight interactions with two cytosolic proteins, calmodulin and immunophilin FK-506 binding protein, proteins that also have been shown to interact with ryanodine receptors. Both calmodulin and FK-506 binding protein appear to have inhibitory effects on channel activity. The IP3 receptor has also been suggested to directly interact with store-operated Ca2+ channels as will be discussed below.

As the three different IP3 receptor subtypes are expressed to various degrees in different cell types, and can often be found in the same cell type, it has been of interest to characterize functional differences that might suggest distinct roles for different subtypes. Differences in Ca2+ sensitivity to open channels have been observed for the type-1 and type-3 receptors, respectively, which have led to the suggestion that the type-3 receptor is ideally suited for providing the first burst of Ca2+ at very low agonist concentrations, while the type-1 receptor may be better suited for regenerating Ca2+ waves.35 Although oscillatory changes in Ca2+ were long known in such excitable tissues as neurons or cardiomyocytes, the advent of intracellular Ca2+-indicating dyes revealed oscillatory Ca2+ waves in many nonexcitable cells. Differential regulation of IP3 receptors by IP3 and Ca2+ are likely to play key roles in generating and maintaining Ca2+ waves.36

Plasma Membrane Ca2+ Channels

In addition to the voltage-gated Ca2+ channels that form the main focus of this volume, three main groups of other plasma membrane proteins allow Ca2+ entry into the cell:

  1. Most ligand-gated cation channels lead to membrane depolarization by providing relatively nonselective passage of both Na+ and K+. However, many of these channels including the neuronal acteylcholine receptors, ionotropic glutamate receptors (in particular the NMDA and AMPA subtypes), and cyclic nucleotide gated channels have significant permeabilities for Ca2+ as well, and Ca2+ influx via such channels has significant physiological effects. We will discuss this in more detail for the cyclic nucleotide-gated channels.
  2. Extracellular ATP acts on cell surface receptors of the P2X and P2Y type. The P2Y type are G-protein coupled receptors and agonist binding often leads to a rise in intracellular Ca2+ via the IP3 pathway. In contrast, P2X receptors are cation channels that lead directly to an increase in intracellular Ca2+.
  3. Store-operated Ca2+ entry (SOCE), also referred to as capacitive Ca2+ entry, is a ubiquitous mechanism that opens a Ca2+ entry pathway in the plasma membrane after intracellular Ca2+ release has depleted intracellular Ca2+ stores. Members of the TRP gene family are considered to be the most likely candidate for SOCE.

Cyclic Nucleotide-Gated Channels

Cyclic nucleotide-gated channels were first described in vertebrate rod photoreceptors. They are found in the outer segment organelles of both rod and cone photoreceptors where the cGMP-gated channels carry the light-sensitive current. Cyclic nucleotide-gated channels have now been found in many different tissues, although their function has only been clearly delineated in sensory cells.37 In retinal photoreceptors, 10-20% of the inward current is carried by Ca2+ under physiological conditions, leading to high sustained Ca2+ concentrations of about 500nM in darkness. Modulation of the cytosolic Ca2+ concentration by light-dependent changes in Ca2+ influx feeds into a powerful negative feedback loop, which involves guanylyl cyclase and mediates the process of light adaptation. Cyclic nucleotide-gated channels are heterotetramers consisting of CNGA and CNGB subunits.

P2X Receptors

P2X gene products are found in many tissues including motor neurons, sensory neurons, airway epithelia, bone, kidney and homopoietic tissue.38 They form fast ATP-gated nonselective cation channels, some of which have a significant Ca2+ permeability. Seven genes coding for P2X receptor subunits have been identified. The P2X subunit proteins range from 384 to 595 residues, and contain 2 transmembrane spanning helices, separated by a large (≈280 residues) extracellular domain. The extracellular domain contains ten conserved cysteine residues and fourteen conserved glycine residues, and two critical disulfide bonds contributing to the ATP binding pocket. Functional P2X receptors are thought to be homo- or heteromultimers, perhaps containing three or six subunits. Two main groups of P2X receptors can be distinguished, those that quickly desensitize (<300 ms) and those that do not or very slowly desensitize. Homomeric P2X7 proteins have the additional property of being permeable to small molecules with a MW of less than 900 kDa.

SOCE and the TRP Gene Family

Agonist-induced rise in intracellular free Ca2+ displays in most cases an initial transient phase due to Ca2+ release from intracellular stores, followed by a more sustained phase that requires the presence of extracellular Ca2+. The latter was initially named capacitive Ca2+ entry, and is now often referred to as store-operated Ca2+ entry or SOCE. Unlike voltage-gated channels, SOCE often shows a high selectivity for Ca2+ over Ba2+ and Sr2+. SOCE has been most convincingly demonstrated with the use of fluorescent intracellular Ca2+-indicating dyes. Characterization of Ca2+ currents induced by store depletion has proven much more difficult, most likely due to the small currents involved and due to low single channel conductance. Such currents have now been described in a limited number of cell types and are referred to as ICRAC currents. Although the molecular entities underlying SOCE have not been resolved unambiguously, mounting evidence suggests that members of the TRP family are involved.39 TRP stands for transient receptor potential referring to a protein in Drosophila photoreceptors that carries a light-activated inward Ca2+ current in the fly visual excitation process. The mammalian TRP gene family contains seven members, most containing about 860 residues. TRP topology is thought to consist of 6 transmembrane spanning helices with a poreforming reentrant loop located between the fifth and sixth helical segments, analogous to the topology of members of the superfamily of voltage-gated channels. The N-terminus is located in the cytoplasm and contains in most cases ankyrin-like repeats as well as a coiled-coiled domain, which may represent sites for interaction with other proteins. The C-terminus is quite variable in length and appears to be more specific for each individual TRP member. Heterologous expression of different members of the TRP gene family has become an important tool to investigate the molecular mechanism of SOCE as will be discussed below.

Mechanisms of Store-Operated Ca2+ Entry

One of the most scrutinized concepts in the study of Ca2+ homeostasis of the past decade is the mechanism by which store depletion leads to Ca2+ entry via the plasma membrane. Although much progress has been made, consensus on unambiguous identification of such a mechanism has remained elusive. Four different mechanisms have been proposed:40-42

  1. A diffusible Ca2+ Influx Factor (CIF) was the first mechanism considered and several partially purified fractions have been reported to activate Ca2+ influx in some cell systems. However, no clear identification has yet been made.
  2. The Ca2+ regulation mechanism proposes that Ca2+ in a restricted space close to the release channel keeps it inhibited; emptying of Ca2+ stores removes the source of the inhibitory Ca2+ and opens the release channel.
  3. The exocytosis model proposes that emptying Ca2+ stores promotes fusion of vesicles containing the Ca2+ release channels with the plasma membrane, thus introducing functional channels to the plasma membrane.
  4. Conformational coupling between the IP3 receptor in the ER with release channels in the plasma membrane.

The latter model has received a lot of attention recently, particularly in cellular systems after transfection with TRP3. Such experiments provide strong evidence for conformational coupling between TRP3 and IP3 receptors. In contrast, elimination of all three IP3 receptor subtypes from a preB-lymphocyte cell line eliminated agonist-induced Ca2+ release, but normal SOCE was observed after store depletion with thapsigargin. As suggested by Putney et al,41 this may indicate that multiple mechanisms exist that couple store depletion to Ca2+ entry, similar to the different mechanisms by which ryanodine receptors are activated in skeletal muscle compared with heart muscle.

Mechanisms to Lower Cytosolic Ca2+

Ca2+ extrusion from the cytoplasm is an energy-requiring process as Ca2+ needs to be moved against a large electrochemical gradient. Two classes of proteins contribute to Ca2+ extrusion from the cytoplasm, those that move Ca2+ at the expense of ATP hydrolysis and those that utilize the energy provided by the inward Na+ gradient to couple Ca2+ extrusion to Na+ influx. Two gene families of ATP-dependent Ca2+ pumps have been identified: the PMCA gene family of pumps found in the plasma membrane and the SERCA gene family of pumps found in the endo- or sarcoplasmic reticulum. Likewise, two gene families of Na+/Ca2+ exchangers have been described: the NCX gene family operates at a stoichiometry of 3Na+/1Ca2+, while the NCKX gene family operates at a stoichiometry of 4Na+/1Ca2++1K+. Thus, members of the NCKX gene family use both the inward Na+ gradient and the outward K+ gradient for Ca2+ extrusion.

PMCA Gene Family

Four members of the mammalian PMCA gene family have been identified, each consisting of about 1200 residues.43 PMCA belongs to the superfamily of P-type ATPases with particularly high expression levels in brain. PMCA operates as an electrogenic Ca2+/H+ exchanger with a 1:1 stoichiometry and a submicromolar affinity for Ca2+. PMCA is thought to be regulated by calmodulin, acidic phospholipids, by neuroactive steroids, and by phosphorylation.

SERCA Gene Family

Three members of the mammalian SERCA gene family have been identified. SERCA1 represents the Ca2+ pump in the SR, expressed mainly in fast-twitched skeletal muscle, and serves the distinction of being the first Ca2+ transport protein for which the crystal structure has been obtained.44,45 Like PMCA, SERCA Ca2+ pumps belong to the P-type ATPases and also consist of a single polypeptide unlike most other P-type ATPases which are heterodimers. SERCA pumps contain ten membrane spanning alpha helices, and three distinct cytosolic domains, the activation domain, the phosphorylation domain and the nucleotide binding domain. SERCA pumps are regulated by a small protein called phospholamban; binding of phospholamban leads to a reduction in the apparent affinity for Ca2+.

NCX Gene Family

Three members of the mammalian NCX gene family have been identified.46 NCX proteins contain about 940 residues and are thought to consist of sets of 5 and 4 transmembrane spanning helices separated by a large hydrophilic loop located in the cytosol. The second set of TM helices is also thought to contain a reentrant loop structure. NCX1 is the dominantly expressed isoform which plays a critical role in Ca2+ homeostasis in heart muscle. Several splice variants have been identified. NCX is a bidirectional Ca2+ transporter and the direction of Ca2+ flux is dependent on the electrochemical Na+ gradient. This exchanger is regulated by secondary interactions with both its substrates, named Na+-dependent inactivation and secondary activation by cytosolic Ca2+. In addition, NCX is activated by phosphatidylinositol-4,5-bisphosphate. NCX1 was the first transporter shown to be regulated by phosphatidylinositol-4,5-bisphosphate; regulation by phosphatidylinositol-4,5-bisphosphate has now been shown for several other channels and transporters including the rod cGMP-gated channel, the PMCA Ca2+ pump and many others.47 Very recently, voltage-gated Ca2+ channels were shown to be regulated by phosphatidylinositol-4,5-bisphosphate as well.33

NCKX Gene Family

Four members of the mammalian NCKX gene family have been identified, which range in size from about 660 residues to 1210 residues.37,46,48 Like NCX, NCKX mediates bidirectional Ca2+ fluxes, dependent in this case on both the transmembrane Na+ and K+ gradients. Although some NCKX isoforms appear to enjoy widespread tissue distribution, very little is known about NCKX physiology outside of the vertebrate retina. In both rod and cone photoreceptors, the NCKX1 and NCKX2 isoforms, respectively, extrude Ca2+ that enters the outer segments of rod and cones via the cGMP-gated channels in darkness. Under bright illumination, NCKX quickly lowers cytosolic Ca2+ and initiates a negative feedback loop that mediates the process of light adaptation. In bovine rod photoreceptors, a rod NCKX1 dimer has been shown to form a complex with the rod cGMP-gated channel heteromultimer.37,49

In summary, calcium ions are of fundamental importance for cell function, and consequently, calcium concentrations are spatially and temporally controlled by a plethora of calcium handling proteins. The remainder of this book will focus in detail on one family of calcium permeant proteins—voltage-gated calcium channels.


This work was supported by an operating grant from the Canadian Institutes for Health Research to P.P.M.S. PPMS is a scientist of the Alberta Heritage Foundation for Medical Research, TGK is a recipient of a studentship from the Alberta Heritage Foundation for Medical Research


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