28.7: Calcium Signaling

Search Fundamentals of Biochemistry

Learning Goals (ChatGPT o3-mini)

Learning Goals: Intracellular Calcium Signaling

By the end of this section, students will be able to:

1. Describe the Unique Chemical Properties of Calcium (Ca²⁺):

   • Explain how Ca²⁺’s low hydration energy, high polarizability, flexible coordination sites, and steep concentration gradient (100 nM intracellular vs. 2 mM extracellular) make it an ideal ion for cellular signaling.

2. Explain Intracellular Ca²⁺ Buffering Mechanisms:

   • Identify the different strategies cells use to maintain low basal Ca²⁺ levels, including sequestration by soluble Ca²⁺-binding proteins (e.g., calmodulin, calbindin, calretinin) and buffering by membrane-associated proteins (channels, pumps, and exchangers).

3. Describe the Storage and Regulated Release of Ca²⁺ in Organelles:

   • Outline the roles of the endoplasmic reticulum, mitochondria, and lysosomes in Ca²⁺ storage.
   • Discuss the functions of key proteins such as SERCA, PMCA, NCX, and TRP channels in transporting and controlling Ca²⁺ movement across membranes.

4. Analyze the Structure and Function of Calmodulin:

   • Illustrate calmodulin’s structure, focusing on its EF-hand motifs and the conformational changes that occur upon Ca²⁺ binding.
   • Explain how calmodulin acts as a Ca²⁺ sensor and transducer to modulate downstream signaling events.

5. Integrate Ca²⁺ Signaling with Other Cellular Pathways:

   • Describe how Ca²⁺ signaling interacts with other pathways (e.g., IP₃/DAG-mediated signaling, store-operated Ca²⁺ entry via STIM and ORAI) to coordinate cellular responses.
   • Explain the role of Ca²⁺ oscillations in modulating the activation of transcription factors (such as NFAT) and kinases (such as CaMKII).

6. Interpret Mathematical Models of Ca²⁺ Oscillations:

   • Understand the basic principles behind models like the Goldbeter model of Ca²⁺ oscillations.
   • Analyze how parameters (such as the rates of Ca²⁺ influx, efflux, and inter-compartmental exchange) affect the frequency and amplitude of intracellular Ca²⁺ spikes.

7. Evaluate the Physiological Significance of Ca²⁺ Dynamics:

   • Discuss how transient rises in Ca²⁺ from low basal levels serve as signals that regulate processes like muscle contraction, secretion, gene transcription, and cell proliferation.
   • Relate alterations in Ca²⁺ handling to disease states and the potential for therapeutic intervention.

These learning goals aim to ensure that students grasp both the molecular details and the integrated systems-level perspectives of Ca²⁺ signaling in cells.

The following is adapted directly and modified from Sharma et al. Biomedicines 2021, 9(9), 1077; https://doi.org/10.3390/biomedicines9091077. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Ca²⁺ is central to numerous cellular processes and functions. Its chemical features, including a low hydration energy, high polarizability, relative flexibility of coordination sites and bond length, and large concentration gradient across cellular membranes (100 nM intracellular to 2 mM extracellular) due to low intracellular levels make it the ion of choice at the core of cellular signaling in prokaryotes and eukaryotes alike.

In studying calcium ion signaling, we will focus on four key areas:

• Buffering of intracellular Ca²⁺ ion concentrations: Basal low levels must be maintained, which allows transient increases to act as signals. Ca²⁺ ions are, hence, no different from other second messengers like cAMP, for example. What matters is the rise from a basal level to a threshold concentration level that allows binding to signaling proteins and subsequent signal transmission.
• Storage of intracellular Ca²⁺: Calcium ions are stored in organelles such as the ER, mitochondria, and lysosomes. The ions must be released in the presence of specific signals and then returned to the storage organelle to maintain basal Ca²⁺ levels.
• Signaling pathways activated by Ca²⁺ ion: We have seen many pathways simulated by a rise in second messengers and by phosphorylation of lipid and protein molecules in interconnected pathways. We will return to several previously studied pathways to see how they integrate with Ca²⁺ in signaling processes.
• Ca²⁺ binding proteins and their binding partners in signaling pathways: We will focus on one key Ca²⁺ binding protein, calmodulin (CAM), and the kinase it activates, Ca²⁺/CAM protein kinases or CAMKs.

The next two sections are adapted and modified from Sharma et al. Biomedicines 2021, 9(9), 1077; https://doi.org/10.3390/biomedicines9091077. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Buffering of intracellular Ca²⁺ ion concentrations

Calcium ions, like the hydronium ion, must be buffered in cells. Otherwise, their potential as a signaling agent would be compromised. The mechanisms adopted by cells for intracellular Ca²⁺ buffering involve sequestration by special proteins, as shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Types of intracellular Ca²⁺ buffers.

Intracellular Ca²⁺ levels are managed through binding to special proteins or sequestration within different cellular compartments. The three main ways intracellular Ca²⁺ is buffered are depicted in Figure 1. These include soluble or unbound proteins that are found in the cytosol or inside organelles, membrane proteins (generally Ca²⁺ channels, ATP-driven pumps (SERCA or PMCA), and ion exchangers (NCX), inside organelles like endoplasmic reticulum (ER), mitochondria, acidic vesicles (mainly lysosomes and Golgi bodies), or organelle junctions (endoplasmic reticulum-plasma membrane (ER-PM), endoplasmic reticulum-mitochondria, or endoplasmic reticulum-lysosomes). The major players regulating inter-organellar Ca²⁺ transfer are IP₃R (inositol-3,4,5-triphosphate receptor), NCX (sodium-Ca²⁺ exchanger), ORAI1/CRACM1 (Ca²⁺ release-activated modulator 1), PMCA (plasma-membrane Ca²⁺ ATPase), SERCA (sarco-endoplasmic reticulum Ca²⁺ ATPase), STIM1 (stromal interaction molecule 1), SOCE (store-operated Ca²⁺ entry), TPC1/2 (two-pore channel), TRP (transient receptor potential), and VDAC (voltage-dependent anion channel). We will discuss some of them below.

These proteins sequester cytosolic Ca²⁺ upon sensing increased Ca²⁺ levels and relay the associated cellular messages. Other proteins that work as intracellular Ca²⁺ buffers exist in the lipid bilayers, plasma membranes, or organelle membranes, such as pumps or transporters. Apart from these proteins, intracellular Ca²⁺ is regulated by inter-organellar transport and the influx of Ca²⁺ ions from the extracellular space.

Storage of intracellular Ca²⁺ - Proteins

Soluble and Unbound Intracellular Proteins: Calmodulin, Calbindin, and Calretinin

Nonmembrane-associated proteins inside a cell can act as both Ca²⁺ sensors and buffers. Most of these proteins have an EF-hand motif that allows Ca²⁺ ions to bind and trigger changes in protein folding, influencing downstream or linked cellular pathways. Calmodulin (CaM) is one of the best-studied and ubiquitously expressed Ca²⁺-sensing proteins known to play a key role in intracellular Ca²⁺ homeostasis. It is a prototype for intracellular Ca²⁺ sensors. It has a 148 amino acid structure with two Ca²⁺-binding sites in two separate lobes, each with two EF-hand motifs. The lobes are connected in the holo (Ca²⁺-bound form that binds other proteins. N- and C-termini alpha-helices with a Ca²⁺ coordination loop in between, providing affinity for Ca²⁺ ion docking and sequestration. The ability of CaM to transmit a change in free intracellular Ca²⁺ levels into a signal depends on the conformational flexibility of the Ca²⁺-dependent (apo) form. CaM can exist in a Ca²⁺-free closed conformational state (Apo-CaM), a semi-open (Ca₂-CaM), or an open state (Holo-CaM or Ca₄-CaM) after Ca²⁺-binding, as shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Overview of calmodulin structure and Ca²⁺ binding conformations.

Figure \(\PageIndex{3}\) shows an interactive iCn3D model of human Holo-calmodulin with 4 bound Ca²⁺ ions (1CLL)

Figure \(\PageIndex{3}\): Holo-calmodulin with 4 bound Ca²⁺ ions (1CLL). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...8nT5HXgNJ4ymc6

Side chains interacting with one Ca²⁺ ion in an EF-hand are shown in the model. The central helix is colored based on hydrophobicity, with green indicating more hydrophobicity. This amphiphilic helix can bind target proteins in this region through nonpolar interactions.

Figure \(\PageIndex{4a}\) shows the change in conformation in the first Ca⁺² site (D20-E31, 1CFD, Xenopus laevis) on binding Ca⁺² (D20-E31, 1CLL, human).  The two Ca²⁺ sites (not the entire protein sequence) were aligned using Pymol in this image.  Carbon atoms are shown in cyan, and the green sphere is Ca⁺². Other atoms are shown with normal CPK colors. 

Figure \(\PageIndex{4b}\): Conformational changes in the first Ca⁺² site (D20-E31, 1CFD, Xenopus laevis) on binding Ca⁺² (D20-E31, 1CLL, human). 

Note the different orientations of the carboxylate side chains as they move to interact with the Ca²⁺ ions.

When applied to all the Ca²⁺ binding sites, these localized conformational changes ultimately lead to large conformational changes in the entire protein.

Figure \(\PageIndex{4b}\) shows the conformational change in the cartoon representation of apo-CAM (cyan, 1CFD, Xenopus laevis) on binding four Ca⁺² ions (magenta, 1CLL, human).  The amino acid sequences of calmodulins from both species are identical.  Both chains were aligned and displayed with Pymol.

Figure \(\PageIndex{4b}\): Conformational changes in the cartoon representation of apo-CAM (cyan, 1CFD, Xenopus laevis) on binding four Ca⁺² ions (magenta, 1CLL, human).

In the holo-form, a fully-formed central helix connects to the two lobes of a "dumbbell-shaped".

Differential Ca²⁺ binding to the two lobes of CaM makes fast buffering of a wide range of free intracellular Ca²⁺ possible for this protein. The presence of methionine residues in its lobes and the plasticity of the central linker in its structure also provide CaM with properties to function as an adaptor protein in intracellular Ca²⁺ signaling. CaM can bind to several targets or effector molecules over a variable distance and in multiple orientations to mediate changes in intracellular Ca²⁺ signaling. Some major effector proteins regulated by CaM binding and relevant for Ca²⁺ homeostasis include EGFR, PI3K, and connexins.

CaM is required for spatial and temporal regulation of [Ca²⁺], as evident by its role in modulating (activating or inactivating) Ca²⁺ pumps (such as PMCA and SERCA) and Ca²⁺ channels (such as CaV1.3, TRPV5 and 6, ORAI). CaM also acts via serine/threonine kinases known as Calmodulin-activated Kinases (CaMKs) to influence cellular processes like proliferation (for example, centrosome duplication at G1/S or anaphase to metaphase transition via CaMKII). We will discuss those in detail below.

Integral Membrane protein molecular buffers: SERCA, PMCA, NCX, and TRP

Integral membrane protein Ca²⁺ buffers primarily translocate free Ca²⁺ between domains and organelles. These mainly comprise ion exchangers, channels, and ATP-driven pumps. SERCA, Sarcoendoplasmic Reticulum Ca²⁺ ATPase, is an ATP-dependent ion pump known to significantly maintain free cytosolic Ca²⁺ concentration via actively pumping the ion into the endoplasmic reticulum (or sarcoplasmic reticulum in muscle cells). They share a general structure that includes 10-pass transmembrane helices and three cytoplasmic domain lobes, as shown in Figure \(\PageIndex{5}\)

Figure \(\PageIndex{5}\): Sarcoplasmic endoplasmic Ca²⁺-ATPase structure (Panel A) and mechanism of Ca²⁺ ion transport (Panel B).

P-type Ca²⁺-ATPases also exist within the plasma membrane and maintain cytosolic Ca²⁺ levels by transferring them into the extracellular space. The Plasma Membrane Ca²⁺ ATPases (PMCAs) can transport one Ca²⁺ ion per ATP molecule, which differs from SERCA's two Ca²⁺ ions per ATP molecule stoichiometry. The general structure of such Ca²⁺ transporters comprises 10 transmembrane segments with large cytosolic loops TM 1–2 and TM 3–4, and cytosolic N- and C-termini tails are shown in Figure \(\PageIndex{6}\), panel A below.

Figure \(\PageIndex{6}\): The general structure of such Ca²⁺ transporters. (A) is the Plasma membrane Ca²⁺-ATPase (PMCA) and (B) shows the Na⁺–Ca²⁺ exchanger (NCX)

The cytoplasmic region of PMCA (left) has three loop structures with binding sites for signaling molecules like CAM. They also have phosphorylation sites for additional regulation. The C-terminal tail contains additional CAM sites as well as a PKA site. It has a PDZ domain that can anchor the protein to cytoskeletal components. Differential RNA processing leads to variations in the amino acid sequence in this region and hence binding specificity. The binding of CaM reverses auto-inhibition of the pump due to conformational shifts, which displace the C-tail from the cytosolic loops. Other means of autoinhibition reversal include phosphorylation of the C-tail (Ser/Thr residues) by protein kinase A or C, proteolytic cleavage of the C-tail, or dimerization via the C-terminus.

Transient Receptor Potential (TRP) channels similarly function in neurons, epithelial, and immune cells. The Mammalian TRP channel superfamily is composed of 28 family members belonging to six subfamilies—TRPC (Canonical), TRPA (Ankyrin), TRPM (Melastatin), TRPV (Vanilloid), TRPP (Polycystin), and TRPML (Mucopilin)—that differ in their sensitivity to various sensory stimulations and affinity for cations (including Ca²⁺ ions) sequestration. Commonly, TRP family members share a structure with six transmembrane domains, intracellular N- and C-termini, and a pore-forming TM 5–6 loop. The cytoplasmic C-terminus of each subunit is a site for protein interaction and post-translational modification. The C-tail of these channels can have PDZ protein binding domains (TRPV and C), sites for interaction with G-proteins (Gq/11)/calmodulin/PLCβ, ADP-ribose binding (NUDIX; TRPM2), or PLC-interacting kinase (PLIK; TRPM6 and 7) domain.

TRP channels act as activators, integrators, and downstream effectors of Ca²⁺ signaling at the plasma membrane and in intracellular compartments. Many members of the TRPC subfamily are activated by DAG (diacylglycerol), which is produced by PLC β- or γ-mediated cleavage of phosphatidylinositol 4,5-bisphosphate (PIP₂) after the ligand binding at GPCRs or RTKs. TRPP1/2, TRPA1, TRPM8, and TRPV1-4 are all expressed on the ER membrane. At this site, PLC-independent activation of the TRP channels (such as TRPV1) is suggested to induce ER Ca²⁺ release via inositol triphosphate receptor (IP3R), further triggering bulk entry of extracellular Ca²⁺ into the cell. On the flip side, cytosolic Ca²⁺ regulates the activity of TRP channels in response to physiological stimuli. This regulatory effect is usually through CaM binding (inhibition of TRPV5, TRPV6, and sensitization of TRPV3) and indirectly through CaM-binding kinase II (CaMKII). These activities are shown in Figure \(\PageIndex{7}\).

Figure \(\PageIndex{7}\): TRP Channels in Signaling. DAG, Diacylglycerol; GPCRs, G-protein coupled receptors; IP₃, inositol 1,4,5-triphosphate; ORAI (Ca²⁺ release activated Ca²⁺ modulator; RTKs, Receptor-tyrosine kinases; ROCE, Receptor-Operated Ca²⁺ Entry; SOC, store-operated Ca²⁺; SOCE, Store-Operated Ca²⁺ Entry; STIM, stromal interaction molecule; PIP₂, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; TRP, transient receptor potential; VDCCs, voltage-dependent Ca²⁺ channels.

Storage of intracellular Ca²⁺ ions - organelles

Endoplasmic Reticulum: STIM, ORAI, IP₃Rs, and TRPC1 in SOCE and SOCIC Ca²⁺ Entry Models

The endoplasmic reticulum (ER) is the largest and most dynamic organelle reservoir for intracellular Ca²⁺ and is therefore central to many signaling processes for protein synthesis, folding, and post-translational modifications. In contrast to the cytosol, ER Ca²⁺ ion levels can range from 100 μM to 1 mM based on the cell type. ER, and other intracellular organelles buffer excessive cytosolic Ca²⁺ by housing Ca²⁺-binding proteins (example: calreticulin in ER) and via active transport (example: SERCA pumps in ER).

• Depletion of Ca²⁺ from the ER lumen activates an indirect mode of Ca²⁺ entry into the organelle, which is called Store-Operated Ca²⁺ Entry (SOCE) or Ca²⁺ Release Activated Ca²⁺ (CRAC) entry. It is activated when plasma membrane receptors like PLC-coupled GPCRs (not voltage-gated channels) trigger Ca²⁺ ion release from the organelle.
• Exhaustion of the intraluminal ER Ca²⁺ ion store following such prolonged release is then sensed by STIM (Stromal Interaction Molecule) tethered to the ER membrane and relayed to the plasma membrane's CRAC channels.

Figure \(\PageIndex{8}\) shows the domain structures of STIM 1/2, ORAI 1-3, and IP3Rs (panel A) and the mechanism of Ca²⁺ influx into the cell (panel B).

Figure \(\PageIndex{8}\): Role of STIM< ORAI and IP3Rs in Ca²⁺ influx.

IP3 Receptors (IP3Rs), on stimulation by IP3, open and allow Ca²⁺ influx from the organelle lumen into the cytoplasm. After activation of IP3Rs on the ER membrane by ligand IP3 and cytosolic Ca²⁺ from activation of phospholipase C, STIM dimers are activated once the luminal Ca²⁺ concentration drops below basal levels. These receptors provide intracellular Ca²⁺ ions for downstream Ca²⁺ signaling, including NFAT-mediated transcription. The red semi-circle in the ER represents high luminal Ca²⁺ levels, the pink semi-circle is for moderately low Ca²⁺ ion concentration, and the pale semi-circle indicates extremely low Ca²⁺ concentration. CC, coiled-coil; NFAT, nuclear factor of activated T-cells; SAM, sterile alpha motif; SOAR, STIM1 Orai1-activating region; TM, transmembrane.

Mitochondria and Acidic Vesicles (Mainly Lysosomes)

Mitochondria also play a critical role in maintaining Ca²⁺ ion levels in the cytosol and endoplasmic reticulum. They are found mostly aggregated around the nucleus and store similar levels of intracellular Ca²⁺ as the cytosol (0.1 μM). Electrochemical proton gradient or membrane potential (Ψ_mt = −150 to −180 mV) and close association to the ER are the key factors responsible for intracellular Ca²⁺ uptake in mitochondria. The free movement of small molecules (less than 5 kDa) from the outer mitochondrial membrane (OMM) into the inner mitochondrial space and their impermeability across the latter generate a high electrochemical proton gradient for ATP synthesis. This gradient simultaneously draws Ca²⁺ ions from the cytosol.

Transfer of Ca²⁺ ions from the ER to mitochondria occurs at specialized microdomains or contact sites known as Mitochondrial Associated Membranes (MAMs). These are characterized by the ER and OMM, which are 10–25 nm from each other and are strewn with a cluster of channels, transporters, exchangers, and tethering proteins for facilitating Ca²⁺ ion transfer. IP3Rs localized at the ER side of the MAMs release Ca²⁺ ions that gate voltage-dependent anion channels (VDACs) on the OMM. VDACs (1, 2, and 3) are 30 kDa polypeptides having a 19-strand beta-barrel structure that regulates the flux of metabolites (polyvalent anions like ADP and ATP) across the outer mitochondrial membranes. These channels transport cations, including Ca²⁺, more readily than anions like chloride. Due to voltage-dependent electrostatic gating, the ion selectivity and flux across VDACs change between open and closed states. Figure \(\PageIndex{9}\) shows coupled mitochondrial and lysosomal effects on intracellular Ca²⁺ signaling.

Figure \(\PageIndex{9}\): Mitochondrial and lysosomal impact on intracellular Ca²⁺ signal.

Primary components of Ca²⁺ signaling at the mitochondrial-associated membranes (MAMs) include IP₃R3 on the endoplasmic reticulum, VDAC1 on the outer mitochondrial membrane, and MCU complex on the inner mitochondrial membrane [151,154,156,161]. Transport of Ca²⁺ ions from the ER to mitochondria plays a crucial role in cellular metabolism (autophagy), cell survival (during unfolded protein response and impinging cell death signals), lipid production, and distribution. 

While IP3 acts as the dominant Ca²⁺-mobilizing messenger, cADPR (cyclic ADP-ribose) and NAADP (nicotinic acid adenine dinucleotide phosphate) are also known to modulate intracellular Ca²⁺ stores. cADPR evokes Ca²⁺ ion release from ER by acting on ryanodine receptors (RyR; counterpart of IP3R in myocytes and co-expressed in some other cell types). NAADP releases Ca²⁺ from acidic and/or secretory vesicles such as lysosomes and endosomes. In most mammalian cells, lysosomes comprise ~5 percent of the cell volume and store similar levels of intracellular Ca²⁺ (0.5 mM) as the ER. Due to their relatively smaller size than ER, lysosomes release nearly undetectable amounts of intracellular Ca²⁺ in response to NAADP trigger.

Signaling pathways activated by Ca²⁺ ions

It is daunting for readers and writers to introduce myriad new signaling pathways. Instead, we will show how Ca²⁺ signaling fits into other pathways we have already discussed. A summary showing how Ca²⁺ signaling integrates with other pathways is shown in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): https://www.creative-diagnostics.com...ng-pathway.htm

An Overview of the Calcium Signaling Pathways

Ca²⁺ signaling, as described above, requires ion buffer, organelle storage, and Ca²⁺ protein pumps and channels. The concentration (amplitude) and frequency of Ca²⁺ release affect signaling. Figure \(\PageIndex{10}\) shows the importance of upstream signaling through GPCRs, phospholipase C, RTKs, and IP3/DAGs. Ca²⁺ also enters the nucleus via IP3 receptors (IP3Rs) and ryanodine receptors (RYR). An important family of cytoplasmic transcription factors, the Nuclear factor of activated T-cells (NFAT), which are important in immune responses and the development of muscle and nervous systems, are activated in calcium ion signaling.

As described above, Ca²⁺ release from the ER is sensed by integral ER membrane proteins called STIMs. These bind Ca²⁺ ions as a buffering system, but the STIM-bound Ca²⁺ ions are also released if most of the ER calcium is depleted. This led to their self-association and ultimate activation of ORAI1, part of the CRAC complex in the cell membranes, which allows extracellular Ca²⁺ ions to flow into the cell in a process called store-operated calcium entry (SOCE). Sufficient calcium now accumulates in the cell to activate the transcription factor NFAT through dephosphorylation by calcineurin (PP2B), also abbreviated CaN in Figure \(\PageIndex{10}\). NFAT then translocates into the nucleus and activates gene transcription. Also, it has been shown that nuclear calcium ions can directly activate the cAMP response element binding protein (CREB), a transcription factor that activates gene transcription. In addition, the CAM:CAMKII complex can translocate into the nucleus. Calcium signals also activate the ERK1/2-MAPK cascade.

Ca²⁺ binding proteins and their binding partners in signaling pathways

We have already described the key calcium-binding protein, calmodulin. On binding Ca²⁺, it undergoes a profound conformational change that allows it to interact with a family of key signaling kinases called CAM and Ca²⁺/CAM-Dependent Protein Kinases (CAMKs).

Ca²⁺/calmodulin-dependent protein kinase (CAMK) is a key signaling protein activated through the actions of Ca²⁺-calmodulin. Activated CAMK is a Ser/Thr kinase. There are many types of CAMKs. We will focus on multifunctional CAMKs that can phosphorylate multiple target proteins. These are important in learning and memory, metabolism, and gene transcription. As with other kinases, they have catalytic and regulatory domains. Some like CAMK II have association domains that allow the formation of CAMKII multimers. In addition, they must have a CAM binding domain. As with all kinases, the CAMKs must be able to switch from an inactive to an active form.

CAMKI has a catalytic, substrate-binding domain and an autoinhibitory domain that blocks the active site. It is activated by an upstream kinase CAMKK (a naming system similar to the MAPK cascade) on the binding of Ca2+ to CAM. CAMKI is found in the cytoplasm and nucleus and helps regulate transcription, the cell cycle, hormone production, cell differentiation, actin filament organization, and neurite outgrowth.

We will focus our attention on CAMKII, which has four isoforms (α, β, γ, and δ). It is activated by the binding of Ca²⁺/CAM, which promotes autophosphorylation. After that, it is active in the absence of CAM. It is important in learning and memory, synapse formation in neurons, and regulating sarcoplasmic reticulum Ca²⁺ transport in skeletal muscles.

They have an N-terminal catalytic domain and a C-terminal association domain that facilitates multimer formation into large holoenzymes with 12 or 14 CAMK monomeric subunits (a homomer or heteromer). These two domains are separated by a linker/regulatory domain with a CAM binding site, an autoinhibitory region, and key Ser and Thr side chains that are targets for phosphorylation. Figure \(\PageIndex{11}\) shows the domain structure of the CAMKII monomer (a), the overall structure of a homododecamer (b), and the mechanism for activation of kinase activity (c).

Figure \(\PageIndex{11}\): CamKII - structure and activation. Zhang, X., Connelly, J., Levitan, E.S. et al. Calcium/Calmodulin–Dependent Protein Kinase II in Cerebrovascular Diseases. Transl. Stroke Res. 12, 513–529 (2021). https://doi.org/10.1007/s12975-021-00901-9. Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/.

T253, T286, and T305/306 are targets of autophosphorylation. M281/282 are also sites for oxidative modification. The C-terminal association domain allows multimer formation. It has a variable region that differentiates CaMKII subtypes. Panel (b) shows the multimer that forms on interactions of multiple association domains on different CAMKIIs. Panel (c) shows that binding of CAM promotes the phosphorylation of key residues, including T286 (through autophosphorylation).

In the absence of Ca²⁺/CAM, the  T286 amino acid forms interactions with the catalytic domain to maintain the inactive conformation. The regulatory domain effectively autoinhibits the kinase domain. On forming the Ca²⁺/CAM/CAMKII complex, a conformation change frees the catalytic domain from autoinhibition and exposes the active site. Each subunit in the dodecamer is activated separately. T286 is now free to be "autophosphorylated" by an adjacent active subunit. Once phosphorylated, pT286 prevents the rebinding of the autoinhibitory region to the catalytic domain, even when CAM dissociates. At this point, CAMKII is active in the absence of Ca²⁺/CAM.

Phosphorylation of T286 also regulates its binding to target proteins for their phosphorylation. In addition, CAMKII can autophosphorylate T254 and T306, which further affects activity. T306 is only autophosphorylated after CAM dissociates, and the enzyme is autonomously active. Dephosphorylation by PP1 and PP2A returns the enzyme to an inactive state.

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of a single subunit of human Ca²⁺ Calmodulin- Dependent Kinase II Holoenzyme (3SOA)

Figure \(\PageIndex{12}\): Single subunit of human Ca²⁺ Calmodulin- Dependent Kinase II Holoenzyme (3SOA). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...S4XAczeqqPE5Y9

The catalytic domain is shown in orange, the association domain in green, and the linker domain in light cyan. The CAM binding site is the spacefill light cyan site in the linker domain. The side chains of Thr 253 and Thr 286, sites for phosphorylation, are shown in spacefill CPK colors and labeled. The side chains of Cys 280 and Met 281, the site for redox regulation, are shown in spacefill CPK colors and labeled. Bosutinib (shown in ball and stick) is an inhibitor of the BCR-ABL and src tyrosine kinase used to treat chronic myelogenous leukemia. It is also an inhibitor of CAMKII, which binds in the catalytic subunit's ATP-binding site. Finally, the activation loop is shown in red.

Figure \(\PageIndex{13}\) shows an interactive iCn3D model of Human Ca²⁺ Calmodulin- Dependent Kinase II Holoenzyme (3SOA)

Figure \(\PageIndex{13}\): Human Ca2+ Calmodulin- Dependent Kinase II Holoenzyme (3SOA). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...DPiWLo5VrgiLp9

The response of most protein kinases we have studied depends on the concentration of a binding ligand (like Ca²⁺). Imagine the ligand modulating the size (amplitude) of the dose-response curve.  The CAMKII dodecamer also responds to the frequency of Ca²⁺ waves or spikes as they are released from intracellular organelles. This is important in neurons where CAMKII phosphorylates ion channels that control and regulate neuron response. If the frequency of Ca²⁺ spikes reaches a certain threshold level, the enzyme no longer depends on Ca²⁺.

Modeling of Ca²⁺ signaling

Model for signal-induced Ca²⁺ oscillations and their frequency encoding through protein phosphorylation.

Reference paper: ALBERT GOLDBETER, GENEVIVE DUPONT, AND MICHAEL J. BERRIDGE , Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 1461-1465, February 1990, Biophysics

In response to hormones and neurotransmitters, cyclic intracellular oscillations/spikes (as a function of time) and spatial waves of cytoplasmic Ca²⁺ arise.  The Ca²⁺ ions derive from opening cell membrane channels and, more importantly, from releasing and recapturing the ions from intracellular compartments such as the endoplasmic reticulum (ER).  Signaling through membrane GPCRs can lead to the activation of phospholipase C and the formation of inositol 1,4,5-trisphosphate (IP3 or InsP₃) as a key second messenger.  IP3 can interact with the IP3 receptor (IP3R) on ER membranes, releasing intracellular stores of Ca²⁺ into the cytoplasm in ways that lead to oscillations in its concentration.   These processes are shown in Figure \(\PageIndex{14}\) below.

Figure \(\PageIndex{14}\): Ca²⁺ oscillations in response to inositol trisphosphate (IP₃) increase, with and without Ca²⁺ influx from extracellular space.  Catacuzzeno L, Franciolini F. Role of KCa3.1 Channels in Modulating Ca²⁺ Oscillations during Glioblastoma Cell Migration and Invasion. Int J Mol Sci. 2018 Sep 29;19(10):2970. doi: 10.3390/ijms19102970. PMID: 30274242; PMCID: PMC6213908.  Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

 

Panel (A) bottom shows a drawing illustrating the hormone-based production of IP₃ that activates the IP₃ receptor (IP3R) to release Ca²⁺ from the endoplasmic reticulum (ER). The biphasic effects of cytosolic Ca²⁺ on IP₃ receptor gating (the basic mechanism for Ca²⁺ oscillations), whereby Ca²⁺ modulates positively the receptor at low [Ca²⁺] but negatively at high [Ca²⁺], are also illustrated. Top, Ca²⁺ oscillations. Note the decaying trend of Ca²⁺ spikes due to the absence of Ca²⁺ influx from the extracellular space.

Panel (B) shows  Ca²⁺ influx apparatus from extracellular spaces through ER-depletion by activated Orai channels on the plasma membrane (bottom), which generates sustained Ca²⁺ oscillations (top). 

Ca²⁺ levels fall through Ca²⁺ pumps on the sarcoplasmic/endoplasmic reticulum (SERCA), which moves the ion into the SR/ER or the plasma membrane (PMCA) that then moves it to the outside of the cell.  The SERCA and PMCA Ca2+ pumps and the ER STIM (Stromal Interaction Molecule that acts as a calcium sensor) proteins are not shown in Figure 14 above.  The STIM1 protein is involved in what is known as store-operated Ca²⁺ entry (SOCE).  This allows the influx of Ca²⁺ after intracellular stores are depleted.  The STIM1 protein has an EF-hand, so it binds and acts as a sensor for Ca²⁺.  When Ca²⁺ is depleted, the STIM1 protein moves from the ER to the cell membrane and activates ORA1, a subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, to promote Ca²⁺  into the cell.

In various cells, hormonal or neurotransmitter signals elicit a train of intracellular Ca²⁺ spikes. The analysis of a minimal model based on Ca²⁺-induced Ca²⁺ release from intracellular stores shows how sustained oscillations of cytosolic Ca²⁺ may develop due to a rise in inositol 1,4,5-trisphosphate (InsP₃) triggered by external stimulation. This rise elicits a certain amount of Ca2+ release from an InsP₃-sensitive intracellular store. The subsequent rise in cytosolic Ca²⁺ triggers the release of Ca²⁺ from a second store insensitive to InsP₃. The model shows how signal-induced Ca²⁺ oscillations might be effectively encoded in terms of their frequency through the phosphorylation of a cellular substrate by a protein kinase activated by cytosolic Ca²⁺.

The release of intracellular Ca²⁺ by IP3 can be broken down into three steps:

• agonist binding to GPCRs to activate the Phospholipase C pathway to produce the second messengers IP3 and DAG;
• IP3 induces some Ca²⁺ release from intracellular stores in the SR/ER.  This can be called the Primer Step, characterized by a rate V1 to produce cytosolic Ca²⁺ of concentration [Z] that primes the next step. This step has a rate of v1β.
• released Ca²⁺ induces more Ca²⁺ release, a self-amplifying positive feedback step, which causes the Ca²⁺ spike. 

A simplified view of this model is shown in Figure \(\PageIndex{15}\) below.

Figure \(\PageIndex{15}\): Schematic representation of the one-pool model based on CICR with Ca2+-stimulated degradation of IP3. Lloyd, C.M., Lawson, J.L., Hunter, P.J., and Nielsen, P.F. The CellML Model Repository. Bioinformatics. 2008 September;24(18):2122-2123 (accessed 4/19/23; 5:40 am EDT).  Creative Commons Attribution 3.0 Unported License.

A simple model can be constructed to account for cytosolic Ca²⁺ fluxes.  The steps (more mathematically defined than described above) include

• IP3 causes a triggering a constant flux of Ca²⁺ into the cytosol, v1β ,to produce cytosolic [Ca²⁺] = Z (the primer)
• cytosolic Ca²⁺ flows into an IP3-insensitive sequestered pool (concentration Y) with rate v2 to keep low levels of cytosolic Ca²⁺  
• Spikes arise when the sequestered pool Y releases Ca²⁺ back into the cytosol at a rate v3, which cytosolic Ca²⁺ activates. 

Goldbeter at al (Proc. Natl. Acad. Sci. 87, 1461-1465 (1990) constructed a mathematical model for the oscillations/spikes. In addition to the parameters mentioned above, they included three more:

• vo, describing the influx of extracellular Ca²⁺ into the cytosol, and k, describing the efflux of cytosolic Ca²⁺ from the cell.  These are controlled by the Ca²⁺ pumps (SERCA, PMCA, et al.) described above. 
• kf, which describes the passive leak of Y into Z.

The Ca²⁺ oscillations are then based on a self-amplified release of Ca²⁺ from intracellular stores. 

Here are their equations:

• \( dZ/dt=v_{0}+v_{1}beta - v_{2}+v_{3}+k_{f}Y-kZ \)
• \( dY/dt=v_{2}-v_{3}+v_{3}-k_{f}Y \)
• \(v_{2}=V_{m2}\frac{Z^{n}}{K^{n}_{2}+Z^{n}} \)
• \(v_{3}=V_{m3}\frac{Y^{m}}{K^{m}_{R}+Y^{m}}.\frac{Z^{p}}{K^{p}_{A}+Z^{p}} \)

We'll use Vcell to plot the following species: 

• Species Z: Concentration (μM) of cytosolic Ca²⁺

• Species Y: Concentration (μM) of Ca²⁺ stored in the InsP₃-insensitive pool. 

The model is based on code from EBI-Biomodels: https://www.ebi.ac.uk/biomodels/BIOMD0000000098.

---

MODEL

Calcium ion oscillation in the cell without oscillations in IP3

Here are the adjustable parameters:

• v0: influx of Ca²⁺ into the cell, uM/s.

• v1: influx of Ca²⁺ into the cell from the InsP₃ receptor, uM/s.

• beta: saturation function of the receptor for InsP₃ , unitless.

• Vm2: maximum rate of Ca²⁺ pumping into the intracellular InsP₃-insensitive store,  uM/s.

• Vm3: maximum rate of Ca²⁺ released from the intracellular store, uM/s;

• K2: Threshold constant for Ca²⁺ pumping, uM.

• n: Hill function cooperativity coefficient for Ca²⁺ pumping into the store, unitless.  

• m: Hill function cooperativity coefficient for Ca²⁺ pumping from the store, unitless.

• Ka: threshold constant for activation, uM.

• kf: efflux of cytosolic Ca²⁺ from the cell, 1/s.

• k:  influx of extracellular Ca²⁺ into the cytosol, 1/s.

• p denotes the degree of cooperativity of the activation process, unitless.

Select Load [model name] below

 Select Start to begin the simulation.

Interactive Element

Select Plot to change Y axis min/max, then Reset and Play  |  Select Slider to change which constants are displayed |  Select About  for software information.

Move the sliders to change the constants and see changes in the displayed graph in real time.

After loading the GoldBeter model, select the 'Start' button below to simulate the model. Adjust the parameter sliders below the plot to see how they affect Ca²⁺ concentrations (Z, Y). The simulator only displays twelve parameters at a time. To choose others, select the 'Slider' button on the side and chose up to twelve parameters to adjust.   

Time course model made using Virtual Cell (Vcell), The Center for Cell Analysis & Modeling, at UConn Health.  Funded by NIH/NIGMS (R24 GM137787); Web simulation software (miniSidewinder) from Bartholomew Jardine and Herbert M. Sauro, University of Washington.  Funded by NIH/NIGMS (RO1-GM123032-04)

---

 

Adjust the parameter sliders below the plot to see how they affect Ca²⁺ concentrations (Z, Y). The simulator only displays twelve parameters at a time. To choose others, hit the 'Slider' button on the side and choose up to twelve parameters to adjust.   

Only two variables, Y and Z, and some intra-connections are required to generate the oscillations, which do NOT require oscillation in the second messenger, IP3. One can imagine that cytosolic Ca²⁺ oscillations might also elicit oscillatory activity of protein kinases activated by it. 

Questions:

• Does v0 change the oscillation frequency?

• What other parameters affect the frequency?

• How does Vm2 affect the oscillations?

Summary

This chapter provides an in‐depth exploration of the central role of calcium (Ca²⁺) in cellular signaling, highlighting its unique chemical properties that make it ideally suited as a second messenger. Key points covered include:

• Unique Properties and Buffering:
  Ca²⁺’s low hydration energy, high polarizability, and flexible coordination chemistry enable it to act effectively as a signaling ion. Despite its high extracellular concentration, cells maintain very low cytosolic Ca²⁺ levels (∼100 nM) through intricate buffering mechanisms involving soluble Ca²⁺-binding proteins and membrane-bound channels, pumps, and exchangers.

• Storage and Release Mechanisms:
  Intracellular Ca²⁺ is stored in specialized organelles such as the endoplasmic reticulum, mitochondria, and lysosomes. Proteins such as SERCA and PMCA actively pump Ca²⁺ into these stores, while release channels (like IP₃ receptors, ryanodine receptors, and TRP channels) enable rapid, transient increases in cytosolic Ca²⁺ upon stimulation.

• Calcium-Binding Proteins:
  Calmodulin (CaM), a prototypical Ca²⁺ sensor, exemplifies how Ca²⁺ binding triggers substantial conformational changes that enable interaction with various downstream targets. Through its EF-hand motifs, CaM translates changes in Ca²⁺ levels into altered activities of enzymes such as Ca²⁺/calmodulin-dependent protein kinases (CAMKs), impacting processes like muscle contraction, metabolism, and gene transcription.

• Integration into Signaling Pathways:
  The chapter emphasizes how the amplitude and frequency of Ca²⁺ oscillations—generated by a balance of release, buffering, and reuptake—encode critical information that regulates cellular responses. These oscillations modulate key transcription factors (e.g., NFAT) and kinases (e.g., CaMKII), integrating Ca²⁺ signals with other pathways such as those initiated by GPCRs and RTKs.

• Mathematical and Computational Modeling:
  Models such as the Goldbeter model are introduced to illustrate how simple coupled reactions and feedback loops can give rise to complex Ca²⁺ oscillations. These models help to elucidate how parameter changes (e.g., rates of Ca²⁺ influx, efflux, and inter-compartmental exchange) affect the dynamics of Ca²⁺ signaling.

Overall, the chapter underscores the importance of precise spatial and temporal regulation of Ca²⁺ in controlling diverse cellular processes and highlights the sophisticated mechanisms that cells employ to harness Ca²⁺ as a versatile signaling molecule.