Calmodulin: a highly conserved and ubiquitous Ca²⁺ sensor

Abstract

Calcium ions (Ca²⁺) play critical roles in various biological phenomena. The free Ca²⁺ concentration in the cytoplasm of a resting cell is at the 10^-7 M level, whereas that outside the cell is 10^-3 M, creating a 10,000-fold gradient of Ca²⁺ concentrations across the cell membrane, separating the intracellular and extracellular solutions.^1),2) When a cell is activated by external stimuli, the intracellular Ca²⁺ concentration increases to levels of 10^-6–10^-5 M through Ca²⁺ entry from the extracellular solution via plasma membrane Ca²⁺ channels and/or Ca²⁺ release from intracellular stores. This transient increase in Ca²⁺ functions as an important signal mediated by Ca²⁺ sensors. Thus, Ca²⁺ signals are transmitted to intracellular loci such as distinct, localized targets of Ca²⁺ sensors. Among numerous Ca²⁺ sensors present in cells, calmodulin is a highly conserved and ubiquitous Ca²⁺ sensor.³⁾

Daybreak of Ca²⁺ studies

The study of Ca²⁺ in biology was initiated by physiologists. S. Ringer first demonstrated the continuous beating activity of frog hearts in the presence of millimolar levels of Ca²⁺ in the blood.⁴⁾ Subsequently, physiologists confirmed that extracellular Ca²⁺ in the millimolar order is required for the secretion of catecholamines from the adrenal medulla and some hormones from endocrine organs.⁵⁾

L.V. Heilbrunn and F.J. Wiercinski showed that microinjection of Ca²⁺ into frog skeletal muscle fibers induced contraction, indicating the involvement of increased intracellular Ca²⁺ in muscle activity.⁶⁾ This finding subsequently influenced biochemists to understand the Ca²⁺-mediated regulation of striated (skeletal and cardiac) muscle contraction. S. Ebashi, at the University of Tokyo, Japan, discovered a protein factor, troponin, that controls Ca²⁺-dependent actin-myosin interactions.⁷⁾ The troponin complex is composed of three subunits: troponin C, troponin I and troponin T. In striated muscle contraction, troponin C is the only Ca²⁺ sensor, which confers a micromolar level of Ca²⁺ sensitivity to actin-myosin interactions via actin-linked regulation.⁸⁾ Before the discovery of troponin C, some Ca²⁺ binding proteins were reported, such as calbindin, parvalbmin, and S-100 protein.⁹⁾ In those days, their biological significance remained unclear. Therefore, troponin C was the first Ca²⁺ sensor for muscle contraction at physiological concentrations of Ca²⁺.

The bridge linking between cAMP and Ca²⁺ studies

In 1958, E.W. Sutherland discovered cAMP as an intracellular messenger.¹⁰⁾ Nowadays, it has been widely accepted as a second messenger involved in many biological phenomena. When ligand-bound, the α-subunit of G-protein coupled receptor (GPCR) dissociates from a βγ dimer, activating the neighboring membrane-bound adenylate cyclase, and resulting in cAMP generation. This increase in cAMP binds and activates protein kinase A (PKA) to trigger physiological processes.¹¹⁾

Since the discovery of cAMP, studies in this field have focused on the regulation mechanism of cAMP levels by generation and degradations. S. Kakiuchi worked in Rall’s lab in the US, particularly in quantifying cAMP in the brain.^12),13) Many researchers competed in the field regarding the regulatory mechanisms of adenylate cyclase, and cAMP generation. After Kakiuchi returned to Japan as the director of the Nakamiya Mental Hospital in Osaka Prefecture, he started to study the control mechanism of cAMP by phosphodiesterase (PDE), involved in cAMP degradation, because this was a less competitive field. The discovery of troponin C, which regulates muscle contraction at micromolar Ca²⁺ concentration levels, had a strong impact on scientists. Kakiuchi was once such scientist who proceeded to examine the involvement of micromolar levels of Ca²⁺ in PDE regulation. However, conducting in vitro experiments at micromolar levels of Ca²⁺ proved challenging because of Ca²⁺ contamination released from glass test tubes, even after washing with distilled water. After repeated trials and failures, he meticulously followed the discovery process of troponin C by Ebashi, and recognized the importance of Ca²⁺-EGTA buffer,¹⁴⁾ which can control the free Ca²⁺ concentration from 10^-7 M to over 10^-5 M. Utilizing Ca²⁺-EGTA buffer, they detected Ca²⁺-dependent and -independent PDE activities in brain supernatants.¹⁵⁾ Ca²⁺-dependent activity was observed below 1 × 10^-5 M of Ca²⁺. From these findings, they discovered a Ca²⁺-dependent PDE-activating factor in a gel- filtered fraction of brain supernatant.¹⁶⁾ This factor was a natural, heat stable protein. They further confirmed that PDE was activated by a protein factor at physiological concentrations of Ca²⁺ (5 × 10^-6 - 2 × 10^-5 M). Thus, these two papers were the first bridge to link cAMP and Ca²⁺ studies. Independently, Cheung in the US reported a protein activator of PDE in brain extract.¹⁷⁾ However, his report did not provide information on the Ca²⁺ sensitivity of PDE activation. Three years later, Kakiuchi and Cheung’s independent research lines converged when J.H. Wang, in Canada, homogeneously purified this protein factor as a Ca²⁺-binding protein.^18),19) This protein was subsequently named calmodulin.

Diverse functions of calmodulin

The early steps in the calmodulin field focused on its primary sequence and structure, then explored its functions, particularly in searching for its targets. Calmodulin was first analyzed by amino acid sequencing. It is a small protein (approximately ～150 amino acids) and its amino acid sequence is highly conserved among species, homologous to that of troponin C.^20),21) Calmodulin has significant homology in and around the Ca²⁺-binding sites, suggesting that Ca²⁺-binding proteins evolved from a smaller ancestral precursor (a single Ca²⁺-binding site) through gene duplication.²²⁾ Calmodulin is widely distributed in various tissues of animals and plants, whereas troponin C expression is restricted to striated muscles. The calmodulin concentration in mammalian cells is estimated to be around 2–10 μM,²³⁾ comparable with the intracellular Ca²⁺ concentration in activated cells. The affinity of calmodulin for Ca²⁺ (Kd = 5 × 10^-7 to -5 × 10^-6 M)²⁴⁾ falls within the intracellular Ca²⁺ concentration range.

Y.S. Babu et al. solved the crystal structure of Ca²⁺/calmodulin,²⁵⁾ and R.H. Kretsinger et al.²⁶⁾ described it as well. Similar to troponin C, Ca²⁺/calmodulin exhibits a dumbbell-like shape. The N- and C-terminal globular domains are connected by an interconnecting α-helix of approximately seven turns. Each globular domain contains two Ca²⁺-binding sites in a helix-loop-helix (EF-hand) structure, indicating that calmodulin and troponin C each possess four Ca²⁺-binding sites per molecule, respectively.²⁷⁾ Conformational changes in calmodulin upon Ca²⁺-binding were further studied using solution techniques, such as nuclear magnetic resonance. By saturating the Ca²⁺-binding sites with Ca²⁺, calmodulin becomes more elongated, suggesting that this elongation upon Ca²⁺ binding might be required for the exposure of hydrophobic side chains to the surface and engaging in hydrophobic protein-protein interactions with target proteins.²⁸⁾ Upon Ca²⁺ binding, calmodulin undergoes a conformational change that actives it ready to bind and influence the enzymatic activity of target proteins.

More than 300 calmodulin-binding proteins (calmodulin targets) have been identified to date (representative examples are summarized in Fig. 1). Among these, calmodulin-dependent enzymes are major targets. Adenylate cycle²⁹⁾ and PDE^15),16) act in opposite directions on calmodulin-dependent enzymes involved in cAMP generation and degradation. Calmodulin regulates these discrepant functions through different intracellular localizations and Ca²⁺ sensitivities. Low Ca²⁺ levels (high-affinity Ca²⁺ binding) through the cell membrane preferentially bind to calmodulin on the cytoplasmic side of membrane-linked adenylate cyclase, resulting in increased cAMP levels, which triggers signal transduction. After cAMP levels reach a maximum, higher Ca²⁺ concentrations (low-affinity Ca²⁺ binding) activate calmodulin-dependent PDE, which rapidly degrades cAMP.³⁰⁾

Fig. 1

Diverse arrays of calmodulin targets. Details are described in the text.

Ca²⁺/calmodulin-dependent protein kinase activity was originally detected in brain synaptosomal membranes.³¹⁾ Gel filtration chromatography was used to separate four groups of Ca²⁺/calmodulin-dependent protein kinases; CaMKI (calmodulin-dependent protein kinase I), CaMKII, CaMKIII (known as specific eEF2 kinase), and CaMKIV.^32),33) Among these, CaMKI, CaMKII and CaMKIV phosphorylate a broad range of substrates, indicating they are multifunctional CaMKs.³⁴⁾ CaMKII accounts for 1–2% of the total protein in the forebrain. CaMKII is highly enriched in neurons, especially in the post-synapse, and plays a critical role in neurotransmission, neuronal plasticity and neuronal network formation.^35),36) In the heart, CaMKII acts as a molecular nexus that is linked to heart failure. Expression of some isoforms of CaMKII and autonomous CaMKII activity are increased in patients with advanced and end-stage heart failure.^37),38) Therefore, the specific inhibition of CaMKII-mediated pathological signaling may be an effective therapeutic strategy for treating heart failure.³⁸⁾ Thus, the main functions of CaMKs are retained for their benefits for human health, and in some cases, CaMKII may be linked to pathological conditions.

Calcineurin is a heterodimer comprising a 60 kDa calmodulin-binding catalytic subunit that interacts with a myristylated subunit of 19 kDa.^39),40) Calcineurin exhibits Ca²⁺/calmodulin-dependent protein phosphatase activity. At the post-synapse, multimeric CaMKII interacts with calcineurin, which is involved in synaptic plasticity.⁴¹⁾ Calcineurin dephosphorylates the nuclear factor of activated T-cell (NFAT) family of transcription factors, causing the nuclear translocation of NFAT and activating gene expression. In T-cells, nuclear-translocated NFAT activates an adaptive immune response.⁴²⁾ The calcineurin inhibitors, cyclosporine A and FK506, are clinically used as immunosuppressants for organ transplantation to prevent organ rejection and autoimmune disorders (atopic dermatitis and psoriasis).^43)-45) However, side effects, such as hypertension, diabetes and hypomagnesemia, are associated with decreased calcineurin function in nonimmune tissues of unknown etiology.⁴⁶⁾

Calmodulin binds to channels and membrane proteins and regulates their function. Calmodulin binding to the NR1 subunit of NMDA receptors in neurons causes a marked reduction in their channel opening probability, suggesting activity-dependent feedback inhibition and Ca²⁺-dependent inactivation of NMDA receptors.⁴⁷⁾ Gap junctions involved in cell-cell communication are composed of connexin family proteins. Calmodulin binds to connexins, resulting in the negative regulation of gap junction channels.⁴⁸⁾ Ca²⁺/calmodulin interacts with the intermediate-conductance KCa channel (IKCa1), which is necessary for the channel to open.⁴⁹⁾ Ca²⁺ gating of small-conduction Ca²⁺-activated K⁺ channels (SK channels) is mediated by Ca²⁺/calmodulin.⁵⁰⁾ Ryanodine receptors are regulated by calmodulin in both the Ca²⁺-free (< 5 × 10^-7 M) and Ca²⁺-bound (> 10^-6 M) calmodulin states. Ca²⁺-free calmodulin enhances affinity of the ryanodine receptor for Ca²⁺, whereas Ca²⁺-bound calmodulin (Ca²⁺/calmodulin) inhibits the channel.^51),52) Calmodulin also interacts with synaptotagmin, which is involved in exocytosis.⁵³⁾

Calmodulin binds to cytoskeletal proteins and regulates cytoskeletal reorganization.⁵⁴⁾ As described previously, troponin C is a critical sensor of the Ca²⁺-dependent regulation of striated actin-myosin interactions via actin-linked regulation.⁸⁾ Despite much effort, troponin complexes (C, I and T) have not yet been detected in smooth muscle or non-muscle tissues and cells. It had been reported that Ca²⁺/calmodulin-dependent myosin light chain kinase phosphorylates the myosin light chain and activates the actin-activated myosin ATPase activity (via myosin-linked regulation) in smooth muscle and non-muscle cells.^55),56) Smooth muscle is a contractile tissue containing abundant contractile proteins, including myosin, actin, tropomyosin and calmodulin. Kakiuchi transferred to Osaka University, where he and K. Sobue began to search for calmodulin targets in smooth muscle and non-muscle tissues⁵⁴⁾ and purified its major target protein, caldesmon, from smooth muscle.⁵⁷⁾ This protein alternately binds to calmodulin or F-actin in a Ca²⁺-dependent manner. At low Ca²⁺ concentration (-10^-7 M), caldesmon binds to F-actin, but not to calmodulin,⁵⁷⁾ and it inhibits actin-myosin interactions. When the Ca²⁺ concentration rises above over 10^-6 M, calmodulin binds to caldesmon⁵⁷⁾ and overcomes this caldesmon-induced inhibition.⁵⁸⁾ Further analysis revealed that smooth muscle caldesmon and its non- muscle isoform inhibit tropomyosin-enhanced actin-myosin interaction, whereas Ca²⁺/calmodulin overcomes this inhibition.^59),60) In these conditions, myosin phosphorylation is a prerequisite for actin-myosin interactions. Ca²⁺/calmodulin and caldesmon control actin-myosin interactions in smooth muscle and non-muscle contractions via actin-linked regulation.^61),62) Thus, smooth and non-muscle contractions may operate via by actin- and myosin-linked dual regulation.⁶²⁾

Microtubules are one of the major cytoskeletal proteins in the brain. Marcum et al. reported the regulation of microtubule assembly in vitro by Ca²⁺/calmodulin.⁶³⁾ We identified the tau protein, a microtubule-associated protein, as a Ca²⁺-dependent calmodulin-binding protein, and it was demonstrated that tau protein-induced microtubule assembly is regulated by Ca²⁺/calmodulin,^64),65) conferring Ca²⁺-sensitivity to microtubule assembly. Afterwards, Miyamoto et al. reported that CaMKII-dependent phosphorylation of the tau protein or MAP2 also regulates microtubule assembly.⁶⁶⁾

In addition to the aforementioned cytoskeletal proteins, calmodulin is also involved in cytoskeleton-mediated events such as motility, morphogenesis, and development including spectrin,⁶⁷⁾ β-adducin,⁶⁸⁾ and myristoylated alanine-rich C kinase substrates (MARCKS).⁶⁹⁾

Certain enzymes contain calmodulin, as a subunit of the enzyme complex. Phosphorylase kinase is a multimeric enzyme (αβγδ)₄ subunit complex, which demonstrates Ca²⁺-dependent activation. The δ subunit is tightly associated with the enzyme, and identified as calmodulin.⁷⁰⁾ Brush border myosin-I (BBMI) is another example of a Ca²⁺-independent calmodulin-target association. BBMI consists of a single myosin heavy chain and three or four strongly associated calmodulin molecules (light chains) without Ca²⁺.⁷¹⁾ However, the significance of calmodulin as light chains in BBMI remain unclear. Nitric oxide synthase (NOS) generates NO, which activates guanylate cyclase, generating cGMP. Three isoforms of NO synthase are present, neuronal NO synthase (nNOS), endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). Constitutive NOS isoforms (nNOS and eNOS) require Ca²⁺/calmodulin, whereas iNOS shows no requirement for Ca²⁺/calmodulin which has calmodulin as a tightly bound subunit.⁷²⁾

Current methods are starting to recognize unexpected arrays of calmodulin targets. Solving the structure of the calmodulin-interacting surfaces to reveal novel target recognition sites may be necessary to uncover the further functions of calmodulin and its targets in health and disease.

Notes

Edited by Shigekazu NAGATA, M.J.A.

Correspondence should be addressed to: K. Sobue, Iwate Medical University, 1-1-1, Idai-dori, Yahaba, Shiwa-gun, Iwate 028-3694, Japan (e-mail: [e-mail]).

Footnotes

This paper commemorates the 100th anniversary of this journal and introduces the following papers previously published in this journal. Kakiuchi, S. and Yamazaki, R. (1970) Stimulation of the activity of cyclic 3′,5′-nucleotide phosphodiesterase by calcium ion. Proc. Jpn. Acad. 46 (4), 387-392 ( https://doi.org/10.2183/pjab1945.46.387); Kakiuchi, S., Yamazaki, R. and Nakajima, H. (1970) Properties of a heat-stable phosphodiesterase activating factor isolated from brain extract: Studies on cyclic 3′,5′-nucleotide phosphodiesterase. II. Proc. Jpn. Acad. 46 (6), 587-592 ( https://doi.org/10.2183/pjab1945.46.587).

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Related Articles

• Kakiuchi, S. and Yamazaki, R. (1970) Stimulation of the activity of cyclic 3′,5′-nucleotide phosphodiesterase by calcium ion. Proc. Jpn. Acad. 46, 387-392.
• Kakiuchi, S., Yamazaki, R. and Nakajima, H. (1970) Properties of a heat-stable phosphodiesterase activating factor isolated from brain extract: Studies on cyclic 3′,5′-nucleotide phosphodiesterase. II. Proc. Jpn. Acad. 46, 587-592.

Appendices

[From Proc. Jpn. Acad., Vol. 46 No. 4, pp. 387-392 (1970)]

Profile

Kenji Sobue was born in Nagoya, Japan, in 1947, graduated from Iwate Medical University, Medical School, and obtained his M.D. in 1973. He entered the Department of Neuroscience at the School of Medicine, Osaka University, as a doctoral fellow, and received his Ph.D. in 1977. He started his academic career as assistant professor in 1977, and then associate professor in 1981, in the same Department at the Graduate School of, Medicine, Osaka University, In 1988, he was appointed as professor of the Graduate School of Medicine, Osaka University. He moved to Iwate Medical University as Vice President in 2011, President in 2016, and CEO in 2023. He received the Young Investigator Award of the Japan Biochemical Society in 1984 and Nakaakira Tsukahara Prize in Japan Neuroscience Society in 1990. His interests focus on the molecular organization of the cytoskeleton and the isolation and regulation of actin-linked proteins, such as caldesmon, spectrin, and other proteins, and also in the cloning of postsynaptic proteins and investigation of synaptic dynamics. Based on these studies, he expanded his interest to neuronal network formation involved in the actin cytoskeleton and their abnormalities in psychiatric disorders.