Biol Res 37: 497-505, 2004

Calcium signaling: A historical account

ERNESTO CARAFOLI

Department of Biochemistry, University of Padova, and Venetian Institute of Molecular Medicine (VIMM), 35121 Padova, Italy

Key words: Calcium signaling, calcium sensors, calcium transporters.

Dirección para Correspondencia

INTRODUCTION: ORIGIN AND DEVELOPMENT OF THE CONCEPT OF Ca² SIGNALING

The idea that Ca²⁺ could be a carrier of signals originated over 100 years ago with the observation by S. Ringer that isolated hearts could only be made to contract if Ca²⁺ was added to the perfusion medium (50). The demonstration that the effect of Ca²⁺ was intracellular and cation specific only came after a long interval of time: it emerged from work performed in 1947 by Heilbrunn and Wiercinski who elicited the contraction of frog muscles by injecting Ca²⁺ into them, whereas no contraction followed the injection of Na⁺, K⁺ or Mg²⁺ (29). While these results unequivocally linked intracellular Ca²⁺ to muscle contraction, the extension of the concept of Ca²⁺ signaling to processes other than muscle contraction was slow in coming. In 1973 Miledi induced neurotransmitter release by injecting Ca²⁺ into the pre-synaptic terminal of squid axons (42), and others elicited responses by injecting Ca²⁺ into mastocytes (33), salivary gland cells (49), or oocytes (61). In parallel with these studies, several reports linked the control of the Ca²⁺ signals to the reversible complexation of the cation by specific proteins, showing that cells contained soluble proteins that buffered Ca²⁺ and, especially, membrane-linked proteins that rapidly and efficiently shifted its concentration within cells by transporting it across membranes. The membrane-intrinsic Ca²⁺-transporting systems in the plasma membrane, mitochondria, endoplasmic reticulum, the Golgi system, and the nucleus, will all be briefly described here, but a succinct discussion of the soluble (or non-membrane intrinsic) Ca²⁺-binding proteins which process Ca²⁺ information will precede the description.

Ca²⁺ BINDING PROTEINS

The most important proteins able to bind Ca²⁺ with the affinity and specificity required for the regulation of its concentration in the intracellular environment belong to the EF-hand family (34), which now contains hundreds of members; some only regulate only one Ca²⁺-dependent process (enzyme), e.g., recoverin, troponin C, while others are not target-specific, e.g., calmodulin, which is the most thoroughly studied EF-hand protein. The structural principles underlying the Ca²⁺-binding function of calmodulin (which corresponds to that of all other proteins of the family) had been originally extrapolated from the crystal structure of parvalbumin (35) but were validated directly by the solution of its three dimensional structure (3). In the structure of EF-hand proteins, domains made of two orthogonal a-helices are interrupted by a 10-12 amino acid loop that coordinates Ca²⁺ to oxygens of some invariant residues. This helix-loop-helix Ca²⁺ binding motif, which is normally repeated several times in the molecules, has been optimized in the course of evolution and it is now found in hundreds of proteins. However, the Ca²⁺ buffering function of EF-hand proteins is secondary to their ability to process the Ca²⁺ signal. The decoding process is performed by two sequential conformational changes (31, 40). The first exposes hydrophobic domains on the surface of the protein upon binding Ca²⁺, enabling it to interact with targets. The second collapses the elongated structure of the protein (calmodulin) around its binding domain in the target enzyme, transmitting to it the Ca²⁺ information.

A quantitative argument must be considered at this point: the total amount of EF-hand proteins (and of other types of proteins that may process the Ca²⁺ signal, e.g., annexins or gelsolin) is finite and could become quantitatively inadequate in cases of large swings in cellular Ca²⁺ concentration. These quantitative restrictions do not apply to membrane-intrinsic Ca²⁺ binding (and transport) proteins, which complex Ca²⁺ at one membrane side, transport it across, and discharge it to the other side, continuously repeating the operation. These proteins thus play the most important role in the buffering of intracellular Ca²⁺, satisfying both the demands for rapid and high-affinity regulation and for lower affinity regulation. For high-affinity Ca²⁺ regulation, cells depend on ATP-driven pumps; for lower affinity regulation, they have more options.

Ca²⁺ TRANSPORT ACROSS MEMBRANES

1. The plasma membrane

The plasma membrane possesses several types of channels which control the downhill diffusion of Ca²⁺ into cells, and two systems that extrude it: a high-affinity, low-capacity pump, and a lower affinity, large-capacity Na⁺/Ca²⁺ exchanger (Figure 1). The plasma membrane is evidently responsible for the 10,000-fold gradient of Ca²⁺ normally measured between the extracellular space and the cytoplasm. However, cells exchange across the plasma membrane only a minor portion of the total Ca²⁺ they need for their activities, using instead for most of their signaling requirements Ca²⁺ stored in the intracellular organelles. Importantly, however, the limited amount of Ca²⁺ penetrating through the plasma membrane triggers cascades of events that are vital to cell activity. Another point on the traffic of Ca²⁺ across the plasma membrane deserves to be mentioned: the very large gradient of Ca²⁺ across the plasma membrane not only ensures unlimited availability of the cation. It is dynamically beneficial, since in its presence even minor changes in the Ca²⁺ permeability of the plasma membrane would ensure significant swings in the intracellular Ca²⁺ concentration. Unfortunately, the large gradient also creates a potential situation of danger. If the Ca²⁺ permeability barrier of the plasma membrane were to fail, which is a frequent event in pathology, the cell would be inundated by Ca²⁺ and its reversible messenger function would come to an end.

Many types of Ca²⁺ channels operate in the plasma membrane: some are gated by ligands (e.g., the channels activated by glutamate in post-synaptic membranes) or by the emptying of the cytoplasmic Ca²⁺ stores (48), but the most important are the voltage-gated channels, which are typical of excitable tissues, e.g. heart. Of the two plasma membrane Ca²⁺-exporting systems, the Na⁺/Ca²⁺ exchanger has low Ca²⁺ affinity but high Ca²⁺ transporting velocity and is particularly active in excitable cells, e.g., heart (see reference 46 for a recent review). The exchanger operates electrogenically, exchanging 3 Na⁺ for 1 Ca²⁺, and thus responds both to the gradients of Ca²⁺ and Na⁺ across the plasma membrane and to the transmembrane potential.

Three basic exchanger gene products are known: NCX1, NCX2, and NCX3. The first two are ubiquitously distributed in tissues, while NCX3 is restricted to brain. They are organized in the membrane with nine transmembrane domains and a large cytosolic loop separating transmembrane domains 5 and 6. The large loop contains regulatory sites, e.g., a putative calmodulin binding domain and an allosteric Ca²⁺-binding site. A particular variant of the exchanger, which is active in retinal photoreceptors, exchanges Ca²⁺ for Na⁺ and K⁺.

Figure 1. The control of cellular Ca2+ Three types of channels gated by ligands, by the emptying of cellular Ca2+ stores, and by voltage mediate Ca2+ entry into cells. The intracellular Ca2+ pool is regulated by binding to Ca2+-binding proteins (e.g., calmodulin) and by Ca2+ transport across the plasma membrane and the membranes of organelles. In addition to buffering Ca2+, soluble Ca2+-binding proteins (e.g., calmodulin) also decode its message. Ca2+ enters the sarco(endo)plasmic reticulum through the sarco(endo)plasmic reticulum Ca2+ ATPase (the SERCA pump) and is released through channels that require effectors like inositol-(1,4,5)-trisphosphate (InsP3) or cyclic ADP ribose (cADPr). The Figure does not show the Ca2+-mobilizing messenger NAADP because the store on which it functions is still undefined. The release route from the sarco(endo)plasmic reticulum creates Ca2+ hotspots, which activate the low-affinity electrophoretic uniporter of neighboring mitochondria. Ca2+ exits mitochondria through a Na+/Ca2+ exchanger (a H+/Ca2+ release exchanger has also been described). Ca2+ can also be exchanged between the cytosol and the nucleus. The nuclear envelope is a genuine Ca2+ reservoir that shares transporters and channels with the ER (not shown in the Figure). The dashed arrows in and out of the nucleus highlight the present debate on Ca2+ traffic through the nuclear envelope (permanent passive permeability versus reversible gating of the pores). Ca2+ is expelled from the cell by a high-affinity/low-capacity plasma membrane Ca2+ ATPase (PMCA pump) and a low-affinity/high-capacity Na+/Ca2+ exchanger. The Golgi vesicles accumulate Ca2+ through a SERCA pump and also through an ATPase that differs from both the SERCA and PMCA pumps and release it through channels of which only that gated by InsP3 has so far been certified.

In animals, the pump is the product of four genes, two (1 and 4) expressed ubiquitously in tissues, two (2 and 3) expressed only in brain and a few other cell types (58). In addition to the four basic gene products, a complex pattern of alternative spicing involving either a domain close to an N-terminal phospholipid interacting site or the C-terminal calmodulin binding domain increases the number of pump isoforms. Of interest are truncated versions resulting from the inclusion of an insert encoded by an extra exon in the C-terminal tail. These truncated isoforms are expected to have lower calmodulin affinity, since the insertion occurs in the middle of the calmodulin binding domain.

2. Intracellular organelles

In the last few years, intracellular organelles—such as the endoplasmic reticulum, the nucleus, the mitochondria, and more recently, the Golgi system—have emerged as crucial to the generation and transduction of Ca²⁺ signals (52). Ca²⁺ in the organelles is important not only in the control of the general Ca²⁺ homeostasis in cells but may also act as a specific regulator of the function of the organelles. A striking example is that of mitochondria, which contain three essential dehydrogenases that are exquisitely sensitive to Ca²⁺.

2.1 The endoplasmic reticulum

Historically, endoplasmic reticulum (or rather, its muscle cell counterpart, sarcoplasmic reticulum, see below) has played the most important role in the development of the concepts of organellar Ca²⁺ homeostasis. The landmark contributions by Hasselbach and Makinose, and Ebashi and Lipmann, in the 1960s showed ATP-dependent Ca²⁺ uptake in muscle microsomal preparations (19, 28). It is now recognized that the maintenance of Ca²⁺ homeostasis within the ER/SR is essential to a number of cell functions, ranging from cell growth, to protein synthesis and folding, to protein processing and transport. The endoplasmic reticulum (ER) and its tissue-specialized species, sarcoplasmic reticulum (SR), are the main dynamic Ca²⁺ storage compartment of eukaryotic cells. They contain a protein pump (SERCA-ATPase) for Ca²⁺ uptake, Ca²⁺ binding proteins for Ca²⁺ storage (calsequestrin and calreticulin), and channels for Ca²⁺ release (the inositol 1,4,5 trisphosphate [InsP₃] receptor, InsP₃R, and the ryanodine- receptor, RyR) (4, 5, 11).

The SERCA pump has 10 transmembrane domains, and its tri-dimensional structure has been recently solved at atomic resolution in both the Ca²⁺-bound and the Ca²⁺-free states (63, 65). The structure has permitted the identification of the location of all important domains, including the ATP binding site and the catalytic aspartyl residue, which are located in a large globular domain protruding between the fourth and fifth transmembrane sectors. All ten transmembrane domains and the cavity that forms the Ca²⁺ binding sites have also been located.

Among the SERCA pumps isoforms, only the cardiac type (which includes the smooth muscle and the slow-twitch muscle isoforms) is regulated by the small hydrophobic protein phospholamban (37) briefly mentioned above. Dephosphorylated phospholamban binds to the SERCA pump, shifting its K_d for Ca²⁺ to a lower affinity value and thus inhibiting the pump activity. Phosphorylation of phospholamban by the cAMP and Ca²⁺/calmodulin-dependent protein kinases dissociates it from its binding site near the active site of the pump (2), relieving the inhibition.

While the InsP₃ that will activate the InsP₃-sensitive Ca²⁺-release channels (InsP₃R) is generated by phospholipase C acting on phosphatidylinositol 4,5-bisphosphate at the plasma membrane in response to a number of first messengers, activation of Ca²⁺ release in ryanodine receptors (RyR) in the terminal cisternal of sarcoplasmic reticulum is triggered directly by plasma membrane depolarization, through direct coupling (i.e., charge transfer) with L-type channels in the T-system in skeletal muscles. However, actual influx of external Ca²⁺ through the T-system L-type channels is instead responsible for the activation for the cardiac RyR. It might be mentioned at this point that, even if they are activated by InsP₃, InsP₃ receptors are also sensitive to Ca²⁺, which thus gates its release from all intracellular channels. Recently, the NAD⁺ metabolite-derivative cyclic ADP-ribose (cADPr), originally discovered as a potent Ca²⁺ mobilizing agent in sea urchin eggs, has been reported to stimulate Ca²⁺ release in higher eukaryotic systems as well. It is likely to act on the ryanodine receptors. A NADP metabolite, nicotinic acid adenine dinucleotide phosphate (NAADP) is the newest addition to the family of activators of intracellular Ca²⁺ channels (23). It discharges a Ca²⁺ pool located in a membrane compartment different from those sensitive to InsP₃ and cADPr (see also Chini, this issue, reference 10).

The genes that encode the InsP₃R and the RyR have now been cloned (21, 44, 60). The InsP₃R/Ca²⁺ release channel is a complex of four subunits (7, 38, 41), organized in a square having a pinwheel appearance with fourfold symmetry (possibly, a channel pore). RyRs were purified from skeletal and cardiac muscles long ago and were also found to be multimeric complexes of very large subunits. Also in this case, electron microscopy and metal shadowing techniques have revealed a fourfold symmetric complex having the shape of a square prism with a bump projecting from the center of one face (32, 66). The number of transmembrane domains in the RyRs is controversial, but it is now accepted that each monomer of the tetrameric InsP₃R contains six transmembrane domains and a large loop between transmembrane domains 5 and 6 which folds within the membrane to form the Ca²⁺ channel (see reference 20 for a comprehensive review).

Within the lumen of the ER, Ca²⁺ is buffered by Ca²⁺-binding proteins, which ensure the storage of large amounts of the cation. Several have been described, among them calsequestrin and calreticulin. These two proteins are not EF-hand proteins: clusters of acidic residues at their COOH-termini are responsible for Ca²⁺ binding.

2.2 The mitochondria

The uptake of Ca²⁺ by mitochondria was postulated by Chance in 1956 based on experiments on the stimulation of electron transport by Ca²⁺ (8), and was experimentally demonstrated by Vasington and Murphy in 1962 (64). Originally, Ca²⁺ handling by mitochondria was simply described in terms of active uptake and passive release (9). Experimental measurements of the internally-negative membrane potential predicted by the chemiosmotic theory (43) across the inner membrane showed that the electrical gradient was the driving force for Ca²⁺ accumulation, which would be mediated by an electrophoretic uniporter (56). It was then discovered that, in addition to the electrogenic uptake uniporter, mitochondria also possess an electroneutral antiporter that extrudes Ca²⁺ from the matrix in exchange for Na⁺ (6). Later on a Ca²⁺:H⁺ exchange reaction was also discovered.

Subsequent work on isolated mitochondria showed that the electrogenic uniporter had low Ca²⁺ affinity and only became activated to appreciable levels when cytosolic Ca²⁺ reached 0.5-1.0 mM (47). Since this concentration was out of the physiological range in resting cells, interest on mitochondria as cytosolic Ca²⁺ regulators gradually faded, shifting the emphasis to the regulation of intramitochondrial Ca²⁺, owing to the Ca²⁺ dependence of some matrix dehydrogenases, i.e., the NAD⁺-dependent isocitrate-dehydrogenase, the 2-oxoglutarate-dehydrogenase, and the phosphatase that activates pyruvate-dehydrogenase (13).

The extrapolation of the results on mitochondrial Ca²⁺ transport to the in vivo situation was prevented by the difficulty of measuring intramitochondrial Ca²⁺ in living cells. Eventually, the targeting of aequorin to mitochondria permitted the reliable measurement of Ca²⁺ in mitochondria in living cells. Mitochondrial calcium spikes occur in response to receptor agonists that promote Ca²⁺ release from endoplasmic reticulum or plasma membrane channel activation, which increase the cytoplasmic Ca²⁺ concentration in the vicinity of mitochondria to the micromolar range, i.e., the concentration required for the optimal activation of the uptake uniporter (51). As a result of these and other findings, interest in mitochondrial Ca²⁺ transport enjoyed a robust renaissance.

2.3 The nucleus

Although most of the traditional targets of Ca²⁺ signaling are located in the cytosol or at the plasma membrane (calmodulin, protein kinase C, ion channels, etc.) recent evidence has indicated that several nuclear processes (the breakdown of the nuclear envelope, transcriptional activation, DNA metabolism, etc.) are modulated by changes of Ca²⁺ in the nucleoplasm (12, 22, 62; also, see references 53 and 54 for recent comprehensive reviews). It has also been shown that nuclear Ca²⁺ may have roles that are distinct from those of cytosolic Ca²⁺, the effects being mediated by the translocation of transcription factors (18) and/or protein kinases (14) into the nucleus. Several reports have shown that nuclear Ca²⁺ activates a number of immediate early genes through the action of calmodulin-dependent kinases (CaMKIV) (25, 27).

2.4 The Golgi

This organelle is the latest addition to the list of Ca²⁺ transporters. It contains a novel P-type ATPase (59) that has extremely high affinity for Ca²⁺ and which accumulates Ca²⁺ into the vesicles. The accumulated Ca²⁺ is released to the cytosol through channels of which so far only that sensitive to InsP₃ has been documented.

CONCLUSIONS

The development of concepts in the area of cellular Ca²⁺ signaling has advanced for six or seven decades in isolated steps separated by long intervals. Originally confined to muscle research, Ca²⁺ as a carrier of information has expanded to other areas. Once the ball started rolling, it all moved at a very rapid pace until Ca²⁺ as a significant agent eventually permeated all fields of biology. Knowledge on Ca²⁺ signaling continues to grow exponentially, enlisting topics such as regulation of gene expression, numerous aspects of neuronal function, the quality control of protein trafficking between membranes, and processes linked to the suffering and eventual demise of cells.

ACKNOWLEDGMENTS

The original work by the authors has been supported by grants from the Italian National Research Council (Targeted Project on Biotechnology), from the Armenise Foundation, from the Italian Ministry of Scientific Research (P.R.I.N. 1998, 2000, 2001, 2002), and from the Human Frontier Science Program (HFSP).

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Corresponding author: Ernesto Carafoli, Department of Biochemistry, University of Padova and Venetian Institute of Molecular Medicine (VIMM) 35121 Padova, Italy. Phone: (39-049) 827-6137, Fax: (39-049) 827-6125, E-mail: ernesto.carafoli@unipd.it

Received: January 26, 2004. Accepted: March 15, 2004.