Elements in Biochemistry The secret life of calcium in cell signalling

Sandip Patel (University Calcium is an abundant alkaline earth metal. In the body, it is found as calcium ions (Ca2+), and most

College London, UK) of it is deposited in hard tissues such as bones and teeth. But less well known is the role of Ca2+ Downloaded from http://portlandpress.com/biochemist/article-pdf/41/4/34/856200/bio041040034.pdf by guest on 30 September 2021 as a messenger within cells. Here, I provide an overview of this hidden but critical function of Ca2+.

The Ca2+ signal: a key message as muscle contraction, secretion and gene expression, to name but a few. Underpinning all of this activity is the The concentration of Ca2+ ions inside the cytosol is crucial Ca2+ signal. Different stimuli evoke Ca2+ increases about the same as outside (1–2 mM). But nearly all of it that can be restricted to a specific part of the cell or is buffered such that the free cytosolic concentration is spread through them and into neighbouring cells as Ca2+ some 10,000-fold lower at ~100 nM. This steep gradient waves. They are often organized as repetitive transients of Ca2+ across the plasma membrane (and organellar (oscillations) of a specific frequency. Cells are tuned to Ca2+ stores) is tapped into during cell stimulation these inhomogeneities in Ca2+ with respect to space and when the cytosolic concentration of Ca2+ rapidly rises, time and therefore act as decoders ensuring precision in entraining a series of events that ultimately influence cell the final outcome. Understanding how Ca2+ signals are function. What is quite remarkable is the diversity of the generated is key to understanding their form and function stimuli that use Ca2+. These include electrical activity and ultimately, their failure in disease. and numerous neurotransmitters and hormones. Equally diverse are the downstream Ca2+-dependent events such Sydney Ringer: the early days

Figure 1. Sydney Ringer. Sydney Ringer (Figure 1) was a clinician at University Credit: Wellcome Collection College London in the 1800s. His research focused (https://wellcomecollection. on defining the constituents of blood that maintained org/works/pvcj26xc). CC BY contraction of the heart. In a classic paper published in the Journal of Physiology, he successfully defined a ‘physiological saline’ that would allow an isolated frog heart to beat for some hours after removal of blood. He correctly deduced that Ca2+ was critical for this. The discovery was fortuitous because in a previous study, he found that he could maintain contractions in a solution containing only sodium chloride. But subsequent experiments failed to reproduce this result and it turned out the former solution was prepared using water that had not been distilled. Analysis of the ‘pipe’ water revealed trace levels of many ions including Ca2+. A systematic investigation using distilled water to which various ions were added uncovered the importance of Ca2+ in contraction. This work laid the foundations for modern research into Ca2+ signalling. Nowadays, solutions based on Ringer’s recipe are widely used both in the lab for experiments and in the clinic to replace fluids.

Ca2+ influx: getting the message across

The extracellular fluid forms an almost infinite reservoir of Ca2+ for signalling purposes. Accordingly,

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Figure 2. Structure of the operated Orai channels. In addition, a super family pore-forming α- subunit of transient receptor potential (TRP) channels and of the voltage gated Ca2+ a number of ligand-gated ion channels are also at our channel, Cav1.1 (green) disposal in both excitable and non-excitable cells to in complex with its mediate Ca2+ influx. These channels respond to various accessory subunits. cues including temperature, noxious stimuli, lipids and neurotransmitters —a rich repertoire of Ca2+-permeable channels allow entry of Ca2+ into cells to evoke Ca2+- dependent processes.

Ca2+ release: an inside job Downloaded from http://portlandpress.com/biochemist/article-pdf/41/4/34/856200/bio041040034.pdf by guest on 30 September 2021 Ca2+ channels are found not only in the plasma membrane, but also on the membranes of intracellular Ca2+ stores, which include the sarcoplasmic reticulum (SR) in muscle cells and the endoplasmic reticulum (ER) in non-muscle cells. They accumulate Ca2+ via ATP- dependent Ca2+ pumps, buffer it through proteins such as calsequestrin and release it through Ca2+ channels

such as IP3 and ryanodine receptors. Unbeknownst to Ringer, although Ca2+ in the extracellular fluid was clearly required for contraction, it was the secondary release of Ca2+ from the SR through ryanodine receptors that sustained the contraction. Ryanodine receptors are Ca2+-activated Ca2+ channels. This means that small increases in cytosolic Ca2+ promote opening of ryanodine receptors resulting in further Ca2+ release in a process referred to as Ca2+-induced Ca2+ release (CICR). In cardiac cells, CICR results from Ca2+ influx through Cav1.2 and type 2 ryanodine receptors (RyR2), which in turn activate additional ryanodine receptors that Ca2+ influx into the cell is a major route for generating ultimately engage the contractile machinery (Figure 3). Ca2+ signals. In the heart, Ca2+ enters through voltage- gated Ca2+ channels. These channels are multi-protein complexes comprising the pore-forming α-subunit and accessory proteins that serve to regulate channel activity and trafficking. Cardiac myocytes predominantly express Cav1.2 α-subunits whereas skeletal myocytes predominantly express Cav1.1, the structure of which has been resolved (Figure 2). When the cardiac membrane depolarizes, critical positively charged residues in each of the four repeat domains that comprise the channel, sense this and transmit conformational changes to the pore region. The pore opens allowing Ca2+ to flow into the cell down its electrochemical gradient. It is this influx that ultimately drives contraction. Similarly, when an action potential arrives at the nerve terminal, it opens voltage-gated Ca2+ channels (Cav2.2). The Ca2+ influx here triggers fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter Figure 3. Excitation-contraction coupling in the heart. thereby propagating the signal across the cleft.

The majority of cell types in our body are non- IP3 receptors form a related family of ubiquitously excitable. Consequently, they rely on voltage-insensitive expressed intracellular Ca2+ channels. They are activated 2+ mechanisms for Ca entry. A major mechanism for by the second messenger molecule IP3 which is produced Ca2+ entry in non-excitable cells is through store- by the phospholipase C family of enzymes in response to

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cell stimulation. Many hormones and neurotransmitters Ca2+ release channels such as certain members of the 2+ activate phospholipase C, evoking IP3 mediated Ca TRP super family and the two-pore channels (TPCs) to signals that go on to regulate numerous processes mediate cytosolic Ca2+ increases. including fluid secretion in secretory cells. Nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca2+ by activating TPCs, which is a Coordinating Ca2+ signals: major way to mobilize lysosomal Ca2+. This is invariably staying connected followed by secondary Ca2+ release from the ER or SR. Here we have an analogy with Ca2+ signalling during Returning to contraction of the heart, we’ve learned excitation-contraction coupling in the heart because that Ca2+ influx through Cav1.2 triggers Ca2+ release by in both cases Ca2+ stores amplify a trigger Ca2+ signal RyR2 on the SR. This communication is facilitated by the deriving from another source. Our work has identified

two proteins being arranged in close proximity where membrane contact sites between lysosomes and the Downloaded from http://portlandpress.com/biochemist/article-pdf/41/4/34/856200/bio041040034.pdf by guest on 30 September 2021 invaginations of the sarcolemma form junctions with ER where such coupling likely takes place (Figure 4). enlarged areas of the SR. NAADP-mediated Ca2+ signals have been implicated in Junctions between different membranes or membrane numerous cellular functions including cell differentiation contact sites feature heavily in Ca2+ signalling. In and muscle contraction. the process of store-operated Ca2+ entry, there is again communication between Ca2+ influx and Ca2+ Ca2+ and disease: combatting Ca2+ release, but here, Ca2+ release triggers Ca2+ influx. Ca2+ communication at membrane contact sites also occurs at Given the multifarious physiological roles of Ca2+, it the interface between the ER and mitochondria. Here, ER is hardly surprising that de-regulated Ca2+ signalling

IP3 receptors are in juxtaposition with the mitochondrial precipitates disease. This is best exemplified by Ca2+ uniporter on the inner mitochondrial to allow channelopathies, i.e., diseases caused by mutations in the coupling Ca2+ release from the ER to Ca2+ uptake by genes encoding Ca2+ channels. Although usually rare, mitochondria which in turn stimulates energy production. they give us valuable information because we can often relate a defined effect of the mutation on Ca2+ channel Ca2+ signalling through acidic organelles: function in vitro to a cellular dysfunction and disease thinking ahead in vivo. Mutations in Cav1.2 in the plasma membrane In addition to the stores of Ca2+ associated with the ER underlie some forms of long QT syndrome, a cardiac and SR, a number of acidic organelles can also serve as disorder. These mutations cause a gain-of-function Ca2+ stores. Chief among the so-called ‘acidic Ca2+ stores’ by preventing channel inactivation. This extends the are lysosomes, which are best known for their role in plateau phase of the cardiac actional potential, which recycling waste material. But over the last decade or so Cav1.2 normally contributes to. This in turn is thought to they have undergone somewhat of a renaissance and are underlie the ensuing arrhythmias. Ca2+ channel blockers now recognized to have numerous roles in signalling. are used clinically to correct arrhythmias in the heart. Like the plasma membrane and ER/SR, they house Defective Ca2+ signals deriving from intracellular sources can also drive disease. Figure 4. Membrane contact sites between Conclusions lysosomes and the endoplasmic reticulum. We’ve come a long way since the classic experiments of Ringer. He would probably be very surprised to learn of the ubiquity of Ca2+ signalling. We know that Ca2+ signals originate from the extracellular space and intracellular Ca2+ stores. In addition, we have built a good molecular understanding of how Ca2+ is handled. But we need to continue to relate this information to health and disease states in order to further understand our biology and to improve on and develop therapeutic strategies.

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Sandip Patel is a Professor at UCL. His Further reading fascination with Ca2+ began during his PhD with Colin Taylor and continued • Berridge, M.J., Lipp, P. and Bootman M.D. (2000) The through postdoctoral work with Andrew versatility and universality of calcium signalling. Nat Thomas and Antony Galione as a Rev. Mol. Cell Biol. 1, 11–21 Wellcome Trust International Travel Prize • Miller, D.J. (2004) Sydney Ringer; physiological saline, fellow. He started his own lab in Oxford calcium and the contraction of the heart. J. Physiol. which he moved to its present location 555, 585–587. in 2001. Sandip’s work has helped develop the concept that • Bers, D.M. (2002) Cardiac excitation-contraction acidic organelles serve as patho-physiologically relevant Ca2+ coupling. Nature 415, 198–205

stores. He is a member of several Editorial boards (including • Wu, H., Carvalho, P. and Voeltz, G.K. (2018) Here, there, Downloaded from http://portlandpress.com/biochemist/article-pdf/41/4/34/856200/bio041040034.pdf by guest on 30 September 2021 Cell Calcium) and Funding agencies (including Parkinson’s and everywhere: the importance of ER membrane UK). He was a former Editor of the Biochemical Journal and contact sites. Science 361, pii: eaan5835 vice chair of the Signalling panel of the Biochemical Society. • Patel, S. and Cai, X. (2015) Evolution of acidic Ca2+ Email: [email protected] stores and their resident Ca2+-permeable channels. Cell Calcium 57, 222–230

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