Intracellular Calcium

By Anthony K. Campbell

Thousands of imaginative scientists, over more than a century, have revealed the fascinating story of intracellular calcium, through a pathway of ingenious invention and discovery. 

Intracellular Calcium, the definitive book on this topic, reveals:

• The pathway of discovery and invention of intracellular calcium over more than 100 years.
• The evidence for intracellular calcium as a universal switch in all animal, plant, fungal and microbial cells
• How the components required for calcium signalling are named and classified.
• The ingenious technology, which has been developed to study intracellular calcium.
• How calcium is regulated inside cells and how it works to trigger an event.
• The role of intracellular calcium in disease, cell injury and cell death.
• How many drugs work through the calcium signalling system.
• How intracellular calcium is involved in the action of many natural toxins.
• How the intracellular calcium signalling system has evolved over 4000 million years, showing why it was crucial to the origin of life.

A key principle presented throughout the book is the molecular variation upon which the intracellular calcium signalling system depends. This variation occurs within the same cell type and between cells with different functions, providing the invisible matrix upon which Darwin and Wallace’s Natural Selection depends.

Featuring more than 100 figures, including detailed chemical structures as well as pictures of key pioneers in the field, a bibliography of  more than 1500 references, as well as detailed subject and organism indices, this definitive work provides a unique source of scholarship for teachers and researchers in the biomedical sciences and beyond.

• Language: English
• Publisher: Wiley
• Release date: Oct 16, 2014
• ISBN: 9781118675533

Book preview

Table of Contents

Title Page

Copyright

Dedication

About the Author

Preface

About the Companion Website

Chapter 1: Setting the Scene: What is So Special About Calcium?

1.1 Discovery of Calcium

1.2 A Natural History of Calcium

1.3 Elements of Life

1.4 Natural Occurrence of Calcium

1.5 Requirement of Cells for Ca²+

1.6 Four Biological Roles of Calcium

1.7 The Puzzle About Ca²+ Inside Cells

1.8 1983 and All That

1.9 Darwin and Intracellular Ca²+

1.10 The Scene Set

1.11 ‘Ja Kalzium, das ist alles!’

Chapter 2: Intracellular Ca2+ – Principles and Terminology

2.1 The Problem

2.2 Some Specific Questions

2.3 Types of Intracellular Ca²+ Signal

2.4 Rubicon Principle

2.5 Key Experiments to Answer Key Questions

2.6 Nomenclature – How Things are Named

2.7 Model Systems

2.8 Darwin and Intracellular Ca²+

2.9 Conclusions

Chapter 3: One Hundred Years Plus of Intracellular Ca2+

3.1 Background

3.2 Why Study the History of Science?

3.3 Tale of Three Pioneers and What Followed

3.4 Ca²+ as an Intracellular Regulator

3.5 Conceptual Development of Ca²+ as an Intracellular Regulator

3.6 Conclusions

Chapter 4: How to Study Intracellular Ca2+ as Cell Regulator

4.1 Pathway to Discover the Role of Intracellular Ca²+ in a Cell Event

4.2 Manipulation of Extra- and Intracellular Ca²+

4.3 Measurement of Intracellular Free Ca²+

4.4 Detecting and Imaging Photons

4.5 Measurement of Total Cell Ca²+

4.6 Calcium Buffers

4.7 Measurement of Ca²+ Fluxes

4.8 How to Study Ca²+ and Other Ion Channels

4.9 How to Discover How the Rise in Cytosolic Free Ca²+ Occurs and Then Returns to Rest

4.10 How to Discover the Intracellular Ca²+ Target and How it Works

4.11 Other Ions

4.12 Conclusions

Chapter 5: How Ca2+ is Regulated Inside Cells

5.1 Principles

5.2 How Resting Cells Maintain Their Ca²+ Balance

5.3 Electrophysiology of Intracellular Ca²+

5.4 Primary Stimuli Which Produce a Cytosolic Free Ca²+ Signal

5.5 Plasma Membrane Ca²+ Channels

5.6 Regulation of Intracellular Ca²+ By, and Within, Organelles

5.7 Second Messengers and Regulation of Ca²+ Signalling in the Cytosol

5.8 Pore Formers and Intracellular Ca²+

5.9 Connexins and Gap Junctions

5.10 Other Ion Channels and Ca²+

5.11 Conclusions

Chapter 6: How Ca2+ Works Inside Cells

6.1 Biological Chemistry of Ca²+

6.2 Ca²+-Binding Proteins

6.3 Ca²+ and Other Intracellular Signals

6.4 Ca²+ and Monovalent Ions

6.5 Transition Metals, Other Divalent Cations and Lanthanides

6.6 Conclusions

Chapter 7: How Ca2+ Regulates Animal Cell Physiology

7.1 Ca²+ and How Nerves Work

7.2 Ca²+ and Cell Movement

7.3 Muscle Contraction

7.4 Chemotaxis and Ca²+

7.5 Intracellular Ca²+ and Secretion

7.6 Ca²+ and Endocytosis

7.7 Intracellular Ca²+ and Intermediary Metabolism

7.8 Intracellular Ca²+ and Cell Growth

7.9 Intracellular Ca²+ and the Immune Response

7.10 Intracellular Ca²+ and Vision

7.11 Intracellular Ca²+ and Other Senses

7.12 Ca²+ and Bioluminescence

7.13 Intracellular Ca²+ and Gene Expression

7.14 Conclusions

Chapter 8: Intracellular Ca2+ and Microorganisms

8.1 The Puzzle

8.2 What Are Microorganisms?

8.3 What Do Microorganisms Do?

8.4 Indirect Evidence of a Role for Intracellular Ca²+ in Bacteria

8.5 Potential Role of Intracellular Ca²+ in Bacteria

8.6 How Much Ca²+ is There in Bacteria?

8.7 How Bacteria Regulate Their Intracellular Ca²

8.8 Ca²+-Binding Proteins in Bacteria

8.9 Regulation of Bacterial Events by Intracellular Ca²+

8.10 Role of Intracellular Ca²+ in Archaea

8.11 Intracellular Ca²+ and Viruses

8.12 Intracellular Ca²+ and Eukaryotic Microorganisms

8.13 Conclusions

Chapter 9: Role of Intracellular Ca2+ in Plants and Fungi

9.1 Role of Ca²+ in Plants

9.2 What Stimulates Plants?

9.3 Requirement of Plants for Ca²+

9.4 Where Ca²+ is Stored in Plants

9.5 Measurement of Cytosolic Free Ca²+ in Plants

9.6 Identification of the Components of the Ca²+ Signalling System in Plants

9.7 How Intracellular Ca²+ Can Provoke Cellular Events in Plants

9.8 Fungal Elicitors

9.9 Apoptosis

9.10 Intracellular Ca²+ and Plant Pathology

9.11 Ca²+ in Mosses, Liverworts and Ferns

9.12 Darwin and Plants

9.13 Ca²+ in Fungi

9.14 Ca²+ and Slime Moulds

9.15 Conclusions

Chapter 10: Pathology of Intracellular Ca2+

10.1 What is Pathology?

10.2 Types of Pathology

10.3 Intracellular Ca²+ – Friend or Foe?

10.4 Intracellular Ca²+ and Cell Death

10.5 Genetic Abnormalities in Ca²+ Signalling Proteins

10.6 Oxygen and Cell Pathology

10.7 Inappropriate Ca²+ Signalling

10.8 ER Stress Response

10.9 Conclusions

Chapter 11: Pharmacology of Intracellular Ca2+

11.1 Background to Compounds That Interact With Intracellular Ca²+ and Ca²+ Movement

11.2 Pharmacological Targets for Intracellular Ca²+

11.3 Drugs Used Clinically That Interfere With Intracellular Ca²+

11.4 Anaesthetics

11.5 Ca²+ Channel Effectors

11.6 Hypertension

11.7 Arrhythmia, Tachycardia and Bradycardia

11.8 Angina

11.9 Heart Failure

11.10 Agents Which Inhibit or Activate Adrenergic Receptors

11.11 Cardiac Glycosides

11.12 Benzodiazapines

11.13 Anti-Psychotic Drugs

11.14 Stimulants and Drugs of Abuse

11.15 Analgesics

11.16 Anti-Depressants and Manic Depression

11.17 Diabetes

11.18 Muscle Relaxants

11.19 Anti-Allergics and Anti-Immune Compounds

11.20 Xanthines

11.21 Substances Used Experimentally to Interfere with Intracellular Ca²+

11.22 Natural Toxins and Poisons

11.23 Plant Toxins and Intracellular Ca²+

11.24 Drugs and the Ca²+ Receptor

11.25 Bacteria

11.26 Ions and Intracellular Ca²+

11.27 Antibodies and Intracellular Ca²+

11.28 Summary and Conclusions

Chapter 12: Darwin and 4000 Million Years of Intracellular Ca2+

12.1 Darwin and Calcium

12.2 Evolution and Ca²+

12.3 What is Evolution?

12.4 Evolution of Ca²+ Signalling

12.5 Darwin and Knock-Outs

12.6 Conclusions

Chapter 13: They Think It's All Over

13.1 What We Know About the Details of Intracellular Ca²+

13.2 What We Don't Know About Intracellular Ca²+

13.3 Intracellular Ca²+ at School and University

13.4 Inspiration of Intracellular Ca²+

13.5 Communicating the Story of Intracellular Ca²+ to Others

13.6 End of the Beginning

Bibliography

Organism Index

Subject Index

End User License Agreement

List of Illustrations

Figure 1

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Figure 1.8

Figure 1.9

Figure 1.10

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

Figure 3.12

Figure 3.13

Figure 3.14

Figure 3.15

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

Figure 4.12

Figure 4.13

Figure 4.14

Figure 4.15

Figure 4.16

Figure 4.17

Figure 4.18

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Figure 6.1

Figure 6.2

Figure 6.3

Figure 6.4

Figure 6.5

Figure 6.6

Figure 6.7

Figure 6.8

Figure 6.9

Figure 6.10

Figure 6.11

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 7.5

Figure 7.6

Figure 7.7

Figure 7.8

Figure 7.9

Figure 7.10

Figure 7.11

Figure 7.12

Figure 7.13

Figure 7.14

Figure 7.15

Figure 7.16

Figure 8.1

Figure 8.2

Figure 8.3

Figure 8.4

Figure 8.5

Figure 8.6

Figure 8.7

Figure 8.8

Figure 8.9

Figure 8.10

Figure 9.1

Figure 9.2

Figure 9.3

Figure 9.4

Figure 9.5

Figure 9.6

Figure 9.7

Figure 9.8

Figure 9.9

Figure 9.10

Figure 10.1

Figure 10.2

Figure 10.3

Figure 10.4

Figure 10.5

Figure 10.6

Figure 11.1

Figure 11.2

Figure 11.3

Figure 11.4

Figure 11.5

Figure 11.6

Figure 11.7

Figure 11.8

Figure 11.9

Figure 11.10

Figure 11.11

Figure 11.12

Figure 11.13

Figure 11.14

Figure 12.1

Figure 12.2

Figure 12.3

Figure 12.4

Figure 12.5

Figure 12.6

Figure 13.1

Figure 13.2

Figure 13.3

Figure 13.4

List of Tables

Table 1.1

Table 1.2

Table 1.3

Table 1.4

Table 1.5

Table 1.6

Table 1.7

Table 1.8

Table 1.9

Table 1.10

Table 1.11

Table 1.12

Table 2.1

Table 2.2

Table 2.3

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 3.5

Table 4.1

Table 4.2

Table 4.3

Table 4.4

Table 5.1

Table 5.2

Table 5.3

Table 5.4

Table 5.5

Table 6.1

Table 6.2

Table 6.3

Table 6.4

Table 6.5

Table 8.1

Table 9.1

Table 9.2

Table 10.1

Table 10.2

Table 11.1

Table 11.2

Table 11.3

Table 11.4

Table 11.5

c01f001

(Reproduced with permission from University of Pennsylvania Archives)

Lewis Victor Heilbrunn (1892–1959)

c01f001

(Endo, 2006. Reproduced with permission from Nature)

Setsuro Ebashi (1922–2006)

Intracellular Calcium

Volume 1

By

ANTHONY K. CAMPBELL

School of Pharmacy and Pharmaceutical Sciences, Cardiff University, UK And Welston Court Science Centre, UK

Title Page

This edition first published 2015

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Library of Congress Cataloging-in-Publication Data

Campbell, Anthony K., author.

Intracellular calcium / by Anthony K. Campbell.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-0-470-69511-1 (cloth)

I. Title.

[DNLM: 1. Calcium—physiology. 2. Biological Evolution. 3. Calcium Channels—physiology. 4. Calcium Signaling—physiology. 5. Cells—drug effects. QV 276]

QP535.C2

612′.01524—dc23

2014004168

A catalogue record for this book is available from the British Library.

ISBN: 978-0470-695111

This book is dedicated to my wife Stephanie.

Thanks for everything.

About the Author

Anthony Campbell was born in Bangor, North Wales, but grew up in London, attending the City of London School. He obtained an exhibition at Pembroke College, Cambridge, and then a first class degree in Natural Sciences and a PhD in Biochemistry at Cambridge University. He moved to Cardiff as a lecturer in Medical Biochemistry at the then Welsh National School of Medicine in 1970, becoming Professor in Medical Biochemistry, followed by Professor in the School of Pharmacy and Pharmaceutical Sciences at Cardiff University (http://www.cf.ac.uk/phrmy/contactsandpeople/fulltimeacademicstaff/campbell-anthonynew-overview_new.html). He has studied intracellular calcium as a cell regulator for over 40 years, pioneering the application of Ca²+-activated photoproteins to measure free Ca²+ in live animal, plant, bacterial and archaeal cells. He is a world authority on bioluminescence, developing the use of genetically engineered bioluminescence to measure chemical processes in live cells. One of his inventions, using chemiluminescence, which is now used in several hundred million clinical tests per year world-wide, was awarded the Queen's Anniversary Prize in 1998, and was selected by the Eureka project of Universities UK in 2006 as one of the top 100 inventions and discoveries from UK Universities in the past 50 years. For the past 15 years his research focus has been lactose and food intolerance, which has led to a new hypothesis on the cause of irritable bowel syndrome, and the mystery illness which afflicted Charles Darwin for 50 years, but was never cured. He is now investigating the relevance of this hypothesis to the current diabetic epidemic, and Parkinson's and Alzheimer's diseases. He has published nine books and over 250 internationally peer-reviewed papers on intracellular calcium, bioluminescence, lactose and food intolerance. Several of his patents have been exploited throughout the world.

Anthony believes passionately in communicating science to the public, and in exciting pupils and students about natural history and cutting edge science. This led him to found the Darwin Centre (www.darwincentre.com) in 1993, now in Pembrokeshire. He also founded the Public Understanding of Science (PUSH) group at Cardiff University in 1994, which organises many events with schools and the public. He has had a laboratory in his house since he was 11 years old. In 1996, he used his patent income to set up the Welston Court Science Centre in Pembrokeshire, which is used as a facility to support the Darwin Centre. He gives regular talks on food intolerance, Darwin, Wallace and bioluminescence, at scientific meetings, to schools and the public. He won the Inspire Wales award for Science and Technology in 2011. He is a Fellow of the Linnean Society and a foreign member of the Royal Society of Sciences in Uppsala, Sweden. In 2013, he was elected a Fellow of the Learned Society of Wales and to the Council of the Linnean Society.

He has been a keen musician all his life, as a tenor soloist, conductor and viola player. Now he is developing a project ‘DNA sings’ to convert light into music. He also makes music in the kitchen – as a keen cook, and has renewed his interest in playing bridge. He has a wife, Stephanie, and five amazing children.

Preface

I keep six honest serving-men

(They taught me all I know);

Their names are What and Why and When

And How and Where and Who.

– Rudyard Kipling, Just So Stories (1902)

The story of intracellular calcium is a marvellous example of how the curiosity of thousands of scientists has led to an understanding of one of the most important regulatory systems in the whole of life – calcium inside cells. This curiosity has catalysed the ingenuity of scientific inventors, who have given us a wide range of molecular, electrophysiological, microscopical and imaging techniques, which have revolutionised biological and medical research. The curiosity about an apparently humble cation, Ca²+, has also led to major breakthroughs in understanding killer diseases, such as heart attacks and strokes, and the consequent development of drugs to treat them. This, quite surprisingly, has produced multimillion dollar markets, with enormous benefits to the world economy and the creation of high-technology jobs. One such example is the remarkable story of a luminous jellyfish, Aequorea, where the curiosity, begun by Osamu Shimomura, about how it produced a green flash when touched, has given us a key indicator for intracellular free Ca²+ and the green fluorescent protein (GFP). Then we have the brilliance of Roger Tsien and the huge contribution he has made, first by inventing a family of fluorescent indicators for intracellular Ca²+, synthesised chemically, and then the genetically engineered Ca²+ indicators based on GFP. The major contribution of Michael Berridge, in the search for the intracellular messenger inositol trisphosphate (IP3) which releases Ca²+ from internal stores, is another example of how scientific curiosity, judgment and persistence can lead to a major discovery. Yet, interestingly, although Osamu Shimomura and Roger Tsien shared the Nobel Prize for Chemistry in 2008, there has been no Nobel Prize for intracellular Ca²+ as such.

Some years ago I gave a lecture about my work at the Karolinska Institutet in Stockholm, Sweden. At an enjoyable supper afterwards, with his group, a member of the Nobel Committee asked me who I thought should win the Nobel Prize for intracellular Ca²+. I was flattered to learn that he had used the first version of Intracellular Calcium: Its Universal Role as Regulator (Figure 1) to make a presentation to the committee. He was very discrete. I said that Roger Tsien and Michael Berridge were obvious candidates. But my actual answer was the two people whose pictures are in the frontispiece. Lewis Victor Heilbrunn was deceased, but Setsuro Ebashi was still alive at the time. His discovery of the first Ca²+-binding protein, troponin C, and the first intracellular Ca²+ store, the sarcoplasmic reticulum, really triggered the explosion in the study of intracellular calcium in the latter part of the twentieth century. The Nobel Prize system is an inspiration to us all. Important as it is to recognise seminal contributions of individuals, the story of intracellular calcium highlights the problem of the prize system. Too many people have made seminal contributions and have made major discoveries. Thank goodness for that, otherwise we might as well all give up!

fpreff001

Figure 1 (a) Intracellular Calcium: Its Universal Role as Regulator (Campbell, 1983). Front cover reproduced with permission from John Wiley & Sons. (b) Rubicon: The Fifth Dimension of Biology (Campbell, 1994).

Campbell, 1994. Front Cover reproduced with permission from Gerald Duckworth & Co. Ltd.

There have been dozens of multiauthor books on intracellular calcium published since my first book, Intracellular Calcium: Its Universal Role as Regulator, was published by Wiley in 1983. In my first book, the aim was to document as well as I could the evidence that intracellular calcium was indeed a universal regulator in living systems. It led me to realise that Ca²+ is both a digital switch and an analogue regulator, depending on the phenomenon concerned. This is the basis of my Rubicon hypothesis (Figure 1). In the present book, my main aim is to explain how Ca²+ actually works inside cells and, crucially, the evidence for this. In particular, I aim to use what we have learnt about the molecular and cellular biology of intracellular calcium, to show why Nature has selected particular components for specific tasks. Why, for example, has muscle chosen to use calsequestrin in the sarcoplasmic reticulum, as its main Ca²+ sink, whereas non-excitable cells such as the liver use calreticulin? Natural history is about describing what goes on in the Universe. Natural science is about understanding how the Universe works. My aim has been to bring together these two essential approaches to scientific endeavour.

To my knowledge there are no other books on intracellular calcium written by one person. Quite a challenge! Multiauthor books provide detailed information on highly focussed topics written by world experts. A single-author book offers the opportunity to develop themes within and between chapters. It also allows the author to develop individual creativity, whilst still retaining the consensus view. Since I was a boy I have had three intellectual passions: a love of nature, natural history; an insatiable curiosity about how nature and man-made things work, natural science; and music, as a tenor, viola player and conductor. This book sings the music of intracellular calcium. Everywhere you look, smell, taste, hear and feel, intracellular calcium is involved. This book is focussed on molecular mechanisms. But, it also aims to focus on the real problems that nature has given us. What really matters is not what happens to an artificial tissue culture cell system in the laboratory, but rather how cells in nature work. Thus, throughout I have addressed the questions about Ca²+ signalling in the natural physiology and pathology of the cells involved. This gives us a great opportunity to enjoy and marvel at the beauties of nature.

I have tried to emphasise two key scientific principles throughout the book. First, to show how intracellular Ca²+ acts as a switch, to activate a wide range of cellular events, and how an analogue mechanism can be superimposed on this digital signalling process, to alter the timing and strength of the cell event. Secondly, in the tradition of Charles Darwin and Alfred Russel Wallace (note his baptism document in the church of St Mary, up the road in Llanbradoc where he was born, shows he was christened Russell with two 'l's because his father misspelt a friend's name), the molecular biodiversity of the components of the Ca²+ signalling system is highlighted, upon which their BIG idea of evolution by Natural Selection critically depends. These themes are a development of two of my previous books (Figure 1). Rubicon: The Fifth Dimension of Biology provided evidence to support the hypothesis that life, throughout 4000 million years of evolution, has depended critically on the evolution of digital events in cells, organisms and ecosystems.

Most importantly, at a cultural level, the story of intracellular calcium has revealed the beauty of molecular biodiversity throughout the animal, plant and microbial kingdoms. Yet, why is this story so poorly dealt with in schools, and even many university curricula? In fact, I have found major mistakes in school exam revision books, including in one physics book – the emphasis on potassium and not calcium in the regulation of the heart beat! As one of the founders of the renaissance, Albrecht Dürer (1471–1528), wrote ‘Be guided by Nature and do not depart from it thinking you can do better yourself. You will be misguided, for truly art is hidden in Nature and he who can draw it out possesses it’. I believe this philosophy is crucial when we teach students at school and university, and when we try to communicate our work to the general public, or even politicians!

There are 13 chapters. Chapter 1 aims to arouse curiosity about what could be special concerning calcium inside cells. Chapter 2 lays down some key principles and identifies important issues about how we name things – nomenclature. Chapter 3 provides an historical overview, starting with Ringer's famous experiments on frog heart at the end of the nineteenth century. Chapter 4 discusses how we can study intracellular Ca²+ and Chapter 5 summarises how Ca²+ is regulated inside cells, so that it can carry out its unique regulatory role. Chapter 6 describes how Ca²+ works in cell and what is unique about the chemistry of intracellular Ca²+. Chapters 7, 8 and 9 deal with the cellular events in animal, microbial and plant cells, which are triggered by a rise in intracellular Ca²+. Chapters 10 and 11 relate to medical and pathological problems, first cell injury and then drugs which affect the Ca²+ signalling system. Chapter 12 is focussed on the evolution of Ca²+ signalling. There is some speculation here. But, hopefully this not too far fetched and, in any event, able to catalyse new thoughts about this fascinating aspect of intracellular Ca²+. The final chapter summarises what we know and what we do not know about intracellular Ca²+. I also discuss the importance of intracellular calcium in the curricula at school and university, and why it is important for professional scientists to engage with schools and the public. We all need to show how curiosity has led to the major discoveries and inventions which have revolutionised all of our lives.

When I give talks to schools or the public I often start by asking the audience what do they think is the greatest gift that evolution has given us? Let's keep sex out of this for a minute! For me, the greatest gift is curiosity. We are the most curious organisms in this planet. I have even been labelled the ‘curious Professor’. No one, except me, is going to read this book from page 1 to the end. Each chapter stands on its own, so there is some repetition between chapters. But I hope by delving into parts you will catch a little of the inspiration I have had from writing it and reading the several thousand references at the end. Please forgive me if I have left out one of your treasured publications. If you feel I have omitted a key paper, or made a mistake, do please email me ([e-mail]), and I will try to add these to a web page and any further editions. The references and other supplementary material will be made available as Endnote files on www.wiley.com/go/campbell/calcium. There will be a student edition - Fundamentals in Intracellular Calcium - to be published 2015/2016, with supplementary material such as hands-on demonstrations for schools and lecturers.

There are many people I'd like to thank. First, my wife, Dr Stephanie Matthews, with whom I have collaborated for over 30 years, and my five wonderful children, David, Neil, Georgina, Emma and Lewis, who have been a great inspiration. My mother died before this book was completed and was a major force in my life. My sister too, Professor Caroline Sewry, who gave me some microscopy pictures for the book. Our dear, late mother Jennet Campbell gave us our musical genes and was amazed that she had produced two science Professors! I have been lucky to have collaborated with many enthusiasts, and to have had many highly able Post-docs and PhD students in my group over the past 40 plus years. Currently, discussions with two colleagues, Ken Wann and Barry Holland, have been vital. Thanks. I thank Barry Holland for his collaboration over 15 plus years and for essential feedback on Chapters 8, Tony Trewavas FRS for feedback in Chapter 9, and Ken Broadley for feedback on Chapter 11. But any errors or omissions are my responsibility. I am particularly grateful to all in the School of Pharmacy and Pharmaceutical Sciences for their tremendous support over the past ten years. I also thank all those who have worked so hard in Pembrokeshire to make the Darwin Centre such a success there, and the many members of my group over 40 years who have helped me investigate intracellular calcium and bioluminescence. Many people at Wiley have worked hard to make this book a success. I thank Paul Deards for his initial enthusiasm for the project, my editor Jenny Cossham, Sarah Tilley Keegan, my first contact at Wiley who has been so supportive and encouraging, and Beth Dufour of RSSP for her vital work on copyright permissions. I would also like to thank Jasmine Kao and Rebecca Lim at Wiley, for their involvement and support with this book. I also would like to thank Ray Loughlin, the copyeditor for the book and Aishwarya Daksinamoorty, Project Manager and her colleagues in SPi Global, who helped craft the various documents into final pages of print which you now see within these covers. I have been lucky enough to have my research funded from a wide range of sources. I thank particularly the MRC, BBSRC (formally SRC and AFRC), NERC, The Wellcome Trust, The Arthritis and Rheumatism Council, The Multiple Sclerosis Society, The British Diabetic Association, The Waterloo Foundation and The Royal Society.

Curiosity inspires, Discovery reveals.

Bon appétit.

Anthony K. Campbell

October 2013

About the Companion Website

This book is accompanied by a companion website. Supplementary material relating to this work can be downloaded at:

www.wiley.com/go/campbell/calcium

The website includes:

Bibliography

Spreadsheets used for equations and calculations

Wav file related to fig 13.4

Table of Contents from Intracellular Calcium: Its Universal Role as Regulator by Anthony Campbell, 1983

Tables in PowerPoint and PDF from Intracellular Calcium: Its Universal Role as Regulator by Anthony Campbell, 1983

References from Intracellular Calcium: Its Universal Role as Regulator by Anthony Campbell, 1983

Chapter 1

Setting the Scene: What is So Special About Calcium?

Ja Kalzium, das ist alles!

–Otto Loewi (1959)

Who would have thought that the serenity of a Bach chorale, the succulent taste of a coq au vin with a nice glass of Côtes du Rhône, the sensuous smell of a flower meadow in spring, the pleasure we get from seeing a puffin as it flies out of its burrow for some more sand eels or even the intellectual excitement of a successful experiment all depend on calcium? This is not the calcium in our diet that most people think of, but rather tiny puffs of calcium inside the cells that are responsible for all our senses, our movements and the functioning of our brain. The fertilisation of our mother's egg by our father's sperm starts life on a wave of intracellular calcium and our embryo then develops on calcium signals within the cells as they differentiate into tissues. We are born on a wave of uterine intracellular calcium, as we are thrust out into the world and start to breathe. Throughout our lives we grow, develop and function through intracellular Ca²+ signals within all of our cells. If we are lucky enough to live until 100, we will have generated over 3000 million puffs of Ca²+ within our heart cells to keep them beating. Finally, we will die on a wave of Ca²+, as it floods into the cells lacking oxygen when our heart eventually stops beating. Changes in intracellular calcium tell a nerve terminal to fire, a muscle to contract and cells to secrete, divide or die. The aim of this book is to explain how changes in calcium inside animal, plant and microbial cells from all the three domains – Bacteria, Archaea and Eukaryota – cause such a wide range of events to occur. A further aim is to show how Darwin and Wallace's BIG idea of evolution by Natural Selection works in real-time, as well as being central to how calcium evolved as a universal intracellular regulator.

There are two defining principles which underpin intracellular calcium as a universal regulator:

Calcium acts as a switch to instruct a cell to cross the Rubicon and do something.

The diversity of molecules that determine when and how a cell fires are a living example of Darwinian variation, upon which Natural Selection depends.

These two principles are dependent on one universal property of cells. All living cells – animal, plant and microbe – maintain a very low free calcium in their cytosol, in the submicromolar range. This results in a huge gradient of calcium across the outer membrane, 10 000-fold in the case of our own cells. It is this calcium pressure that has allowed evolution to capitalise on the unique chemistry of calcium for it to act as a cellular switch.

1.1 Discovery of Calcium

Humphry Davy (1778–1829; Figure 1.1) was one of the founders of modern chemistry. Working at The Royal Institution in Albemarle Street, just off Piccadilly in the centre of London, he came up with an idea that lies at the heart of understanding biological chemistry. He proposed that what we now call salts were made up of two parts: one positive, the other negative. He realised that there was an obvious way to test this hypothesis – connect a salt solution to a battery and this would separate them.

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Figure 1.1 Sir Humphry Davy, Bt, FRS, MRIA, FGS (1778–1829).

Sir Humphry Davy, by Thomas Phillips, 1821 who discovered calcium in 1808. Courtesy of the Royal Institution.

Davy was born in Penzance in Cornwall, United Kingdom, on 17 December 1778. His father, Robert Davy, was a wood carver at Penzance, who pursued his art rather for amusement than profit and was the representative of an old family dating back as far as 1635. Davy's mother, Grace Millet, also came from an old but no longer wealthy family. Davy was a precocious boy, with an amazing memory. His interest in scientific experiments began when he was less than 10 years old. After the death of his father in 1794, Davy began an apprenticeship with a local surgeon John Bingham Borlase, soon becoming a fully qualified chemist. Then, in 1798, Davy joined the ‘Pneumatic Institution’ at Bristol, established to investigate the medical powers of ‘factitious airs and gases’. This was the opportunity for Davy to begin his experimental career in earnest.

In 1799, Count Rumford had proposed the establishment in London of an ‘Institution for Diffusing Knowledge’. Thus, the Royal Institution was founded, its building in Albemarle Street being set up in April 1799. Rumford was the first secretory to the institution and a Dr Garnett was the first lecturer. But it was Joseph Banks, founder of Kew Gardens and a President of the Royal Society, who had recognised Davy's remarkable talents as a scientist and recommended Davy to move to the Royal Institution. He did so in 1801, and it was Davy, in the years that followed, who made it famous for its scientific discoveries and for communicating science to the public. Davy's lectures included spectacular and sometimes dangerous chemical demonstrations for his audience. He also discovered nitrous oxide, and its anaesthetic and hallucinogenic properties, which he introduced to many distinguished guests. In 1804, Davy became a Fellow of the Royal Society, becoming its President in 1819. In the same year, Davy was awarded a baronetcy – at that time the highest honour ever conferred on a British scientist.

In 1807, he began to test his hypothesis that salts were composed of positive and negative components by putting a voltage across a solution of potassium hydroxide (Davy, 1807). The experiment failed, as all he succeeded in doing was to electrolyse water. He then read of some pioneering experiments by two Swedish scientists, Jöns Jacob Berzelius (1779–1848) and Magnus Martin Pontin (1781–1858), who lived together in Stockholm, carrying out chemical experiments in their kitchen. They discovered that they could use mercuric oxide, which Joseph Priestley (1773–1804) had used to discover oxygen, to trap some elements as a mercury amalgam. So Davy decided to reconnect a large battery he had made across molten potash (KOH) adding also mercury. On recovering the mercury amalgam that formed at the negative electrode, he distilled off the mercury to reveal his first alkali metal, potassium. This was so reactive in air that it caught fire. His brother later described Davy's excitement as he rushed around the room, spilling highly corrosive nitric acid all over his clothes! A year later he tried the same experiment, this time using a moist mixture of lime and red oxide of mercury, but again with mercury at the negative electrode (cathode). This time removal of the mercury by distillation revealed a new metal, ‘greyish-white with the lustre of silver’. This also burnt avidly in air with a brick-red flame to form the nitride and some oxide (lime). Davy called this metal ‘calcium’ after the Latin for lime – calx. He reported these findings to The Royal Society on 30 June 1808 (Davy, 1808a). Davy wrote perceptively that electricity was to be of great value in discovering ‘the true elements’ (Davy, 1808b). It is the ability of Ca²+ to bind reversibly to particular negatively charged ions (anions), together with its ability to carry current, which holds the key to understanding the unique role that calcium plays throughout the animal, plant and microbial kingdoms. Davy went on to use electrolysis to discover sodium, magnesium, barium and boron.

Davy appointed a laboratory assistant, Michael Faraday (1791–1867), who enhanced Davy's work, eventually becoming the more famous and influential nineteenth century scientist. This led to Davy claiming Faraday as his greatest discovery, but later accusing Faraday of plagiarism, causing Faraday (the first Fullerian Professor of Chemistry) to stop his pioneering experiments on electromagnetism, which eventually led to the invention of the dynamo and the electric motor, until his mentor's death.

In 1815, Davy invented a lamp which allowed miners to work safely in the presence of methane. The Northumberland miners had come to London in order to meet the best chemist in England, in the hope that he could prevent the large number of deaths in their mines as result of ‘fire damp’ (methane) exploding. Davy, with his protégé Faraday, set about trying to find a way of controlling the reaction of methane with oxygen. They discovered that copper mesh would allow a methane flame to flare up without exploding, because the holes were too small to let a flame through. The Davy lamp was born. It did not prevent an explosion, but did act as an indicator that the methane level in a mine was too high, giving time for the miners to escape the explosion. The first trial of a Davy lamp with a wire sieve was at Hebburn Colliery on 9 January 1816. Over 150 years later, chemical indicators for calcium, which Davy had discovered as an element, were to be critical in establishing its universal role as an intracellular regulator.

Davy had a reputation of a somewhat irritable temperament, but was always highly enthusiastic and energetic. Davy died in Switzerland in 1829, attempting to recover from a heart condition, thought to be inherited from his father's side of the family. During the last few months of his life, Davy wrote a book incorporating poetry, scientific philosophy and speculations about alien life. This was popular for several decades amongst the Victorian intelligentsia. Davy is buried in the Plainpalais Cemetery in Geneva. Although his death is attributed to heart disease, there has been some speculation that he suffered long-term consequences as a result of his frequent experimental procedure of distilling mercury and his playing with nitrous oxide.

Davy and Faraday, through the Royal Institution, were pioneers in demonstrating scientific discoveries by experiment to the general public. Some lectures were so popular and were attended by visitors such as Coleridge, that Albemarle Street was made a one-way street for carriages, the first in the world! Today, most people are familiar with the importance of calcium in bones and teeth. Yet few are aware of the central role calcium plays inside all the cells of the body. Intracellular calcium hardly features at all in the school science curriculum. This is a topic I shall address in the last chapter. Many of us believe that academics must reach out and engage with the public to explain why we do what we do and to excite the next generation about how great it is to follow a lifetime of curiosity through science. I established the Darwin Centre in Wales (www.darwincentre.com) for this purpose, which, in the tradition of naturalists like Darwin, takes descriptions of natural history into the mechanisms of natural science (see Chapter 13). It is this philosophy which I will try to follow throughout this book.

1.2 A Natural History of Calcium

Calcium was one of the earliest elements to form in the Universe and is now 13th in order of abundance, hydrogen being the first (Mason, 1991; Tegethoff, 2001). In the Earth's crust, calcium is the fifth most common element and the third most common metal element, being twice as abundant as magnesium (Figure 1.2). Everywhere you look there are examples of calcium precipitates outside cells – in rocks, in the cement holding buildings together, in the sea and every time someone smiles at you. But this book focuses on a quite different role of calcium – how it regulates physiological and pathological processes within living cells. So, let us examine two scenarios to help us focus on this central theme – how calcium plays its unique biological role as a universal regulator inside all living cells.

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Figure 1.2 The relative abundance of elements in the Earth's crust. Data expressed relative to hydrogen as a molar ratio or as parts per million (ppm) (converted from Day, 1963; Fairbridge, 1972; Fyfe, 1974). Hydrogen = 1460 moles per million grams. Relative abundance in many publications is calculated on a weight basis as ppm. But molar ratios relative to hydrogen are more relevant to the chemical reactivity of the element concerned. The data here applies only to the Earth's crust. In the Earth's mantle, magnesium, for example, is at least 30 times as abundant as calcium.

Reproduced by permission of Welston Court Science Centre.

1.2.1 Calcium by the Sea

Wales, where I live, has some of the most beautiful coastline in Europe, with estuaries, sandy beaches, cliffs and coastal paths with superb views. There are small islands off shore packed with wonderful wildlife, both above and below water. But most important of all, we have fantastic tides. Just 100 m from my house is a beach with the second biggest tidal fall in the world, over 12 m on many spring tides – that's upwards! This means we get some of the best shore marine life and rock pools in the world. So imagine you are on holiday glancing into one of these rock pools, with its abundance of animal and plant life (Figure 1.3).

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Figure 1.3 Calcium by the sea: some organisms easily found on rocks or in rock pools when the tide goes out.

Reproduced by permission of Welston Court Science Centre.

The rock itself is made of limestone – calcium carbonate deposited millions of years ago by extinct calcified organisms. The sea water within the pool contains calcium at a concentration over 10 times that free in the blood. Within the pool are examples of calcium outside cells. On the fringe, there will be tufts of the pinkish seaweed Corallina covered in a secretion of calcium carbonate (CaCO3). On the floor of pool there are the skeletal remains of a dead fish, made of calcium phosphate. And the surrounds of the pool, in and out of water, are littered with the ‘sheep’ of the rocks – limpets, whelks and periwinkles grazing on small algae, as well as mussels and barnacles, all exhibiting their ability to form hard shells from calcium carbonate. But the mussels and barnacles are also revealing another role for calcium, inside cells.

The mussels and barnacles under water in the rock pool itself are open, showing their fine antennae filtering out small planktonic organisms to feed on. But those out of water are firmly shut! They are closed because a calcium signal in their large muscle fibres has caused these to contract, keeping their soft insides protected from drying out in the sun. Underneath one of the rocks there is a sea urchin with a cluster of its eggs, ready to develop because a calcium wave has been triggered by the sperm that fertilised them. A sea anemone clutches your finger, trying to anaesthetise you by injecting its poison from the tiny syringes in its sting cells (nematocysts) triggered to fire by a calcium signal inside each cell. The toxin in its sting works by affecting cation channels in your sensory nerves. Also stuck on the underside of the rock is a sea slug, which has synchronised electrical signals provoked by calcium moving into the cell through special ion channels. There are fish and shrimp darting about, whose movements are all regulated and coordinated by small puffs of calcium in their muscle cells, and at the terminals of all their nerves. And then you notice a tiny carpet of what look like small flowers attached to a piece of kelp – a seaweed attached to the side of the pool. These flower-like organisms are in fact animals – the hydroid Obelia geniculata, part of the life cycle of a small jellyfish. Obelia flashes blue-green light when touched in the dark. This is caused by a chemical reaction within an intracellular protein, obelin (Campbell, 1974a, b). The protein flashes when it binds calcium. It was its relative, aequorin, from an American jellyfish, that provided the first universal method for the direct measurement of free calcium inside a living cell (Ridgway and Ashley, 1967; Ashley and Ridgway, 1968). The jellyfish Aequorea was also the organism that gave us green fluorescent protein (GFP) (Shimomura et al., 1962; Shimomura et al., 1963; Morin and Hastings, 1971b) that has had such an impact on cell biology, enabling a range of calcium and other intracellular indicators that change colour to be genetically engineered (Miyawaki et al., 1997; Waud et al., 2001). Even the microscopic cyanobacteria, vital in global warming, that are coating some of the animals and plants growing on the side of the pool, can use calcium to regulate their ability to fix nitrogen.

Calcium is everywhere to be seen, inside and outside of the cells that make up the organisms in the rock pool (Table 1.1). Within the limestone sides of the pool there is a fossil ammonite, some 200 million years old, showing us that calcium has been important in life throughout millions of years of evolution. As you clamber up the cliff back to your bike, you get a good view of the bushes and small trees, shaped by the prevailing wind. These have not been mechanically blown into these streamlined shapes, but rather signalled to grow that way. Repetitive, tiny puffs of calcium have been triggered inside the cells day by day as a defence against the wind, causing the cells on one side of the bush to grow at a different rate from those on the other side.

Table 1.1 Roles of calcium by the sea.

1.2.2 Calcium in Your Wake-Up Call

The alarm clock rings. It's 7 a.m. Your eyes open, your heart starts to race a little and you jump out of bed. Last night was one to remember! After a quick shower, you munch a piece of toast and marmalade, and quickly drink your usual cup of tea. You rush out of the door to work. You have a lecture on cell signalling, which starts promptly at 9 a.m. As you jump on your bike, a thought crosses your mind. Without little puffs of calcium inside the cells of your brain, heart, leg muscles, pancreas and liver you wouldn't have been able to wake up and get out of bed, let alone digest your breakfast (Table 1.2). And without Ca²+ puffs in your parent's gametes you wouldn't even have been conceived! Yet the timescale, strength and nature of the Ca²+ signal, and the event itself, vary incredibly with cell type.

Table 1.2 Calcium gives you a wake-up call: examples of how intracellular Ca²+ is crucial in enabling you to wake up and get to work.

The timescale over which intracellular Ca²+ works varies with cell type from milliseconds to hours. Thus, intracellular Ca²+ triggers a nerve cell to release its neurotransmitter in milliseconds and the Ca²+ signal that induces a heart beat lasts just 1 s. In contrast, the secretion of insulin and digestive enzymes from the pancreas, together with the stimulation of intermediary metabolism in muscle and liver after breakfast, last minutes or even an hour or so. Yet a mussel or barnacle stranded at low tide has to use its muscles to keep its shell shut for several hours until the tide comes back in again. The regulation of the cell cycle by Ca²+ may take days or even weeks to take effect through regulation of gene expression. Diseases such as cancer or Alzheimer's, where intracellular Ca²+ may be involved, can take months or years to show up.

The calcium signalling system is now a prime target for drug discovery in controlling heart disease, blood pressure, diabetes, arthritis, multiple sclerosis, cancer, diseases of the brain and nervous system, several genetically based diseases, and potentially many infections by bacteria and viruses. Calcium even has a major role in keeping oxygen and nitrogen in the atmosphere at the right level, and in the microorganisms that are involved in controlling global warming.

This then is the fascinating puzzle about calcium. How does one simple cation do all of this?

The answer will depend crucially on the source of the Ca²+ for the intracellular change, whether it is internal, external or both, and what type of Ca²+ change occurs. Is it a single cytosolic Ca²+ transient? Does it form a wave or tide of Ca²+ as the cell fills up with Ca²+? Or does it involve a series of Ca²+ spikes or oscillations to maintain Ca²+ bound to its target for many minutes or hours, without Ca²+ draining out of the cell? It will also depend on the location and the type of Ca²+ target in the cell.

So these scenarios highlight that there is something very special about the chemistry of calcium inside living cells. Evolution has selected Ca²+ as the universal intracellular switch for an amazing variety of phenomena in animals, plants and microbes. Abundant monovalent cations like Na+ or K+, or divalent cations such as Mg²+, Cu²+ or Zn²+, just did not have the right chemistry for the task in hand.

1.3 Elements of Life

During nearly 4000 million years of evolution, living systems have exploited the chemical and electrical properties of 29 elements – 16 metallic and 13 non-metallic (Table 1.3). Metals always work as cations (positively charged ions). These are responsible for maintaining the osmotic balance of cells, their electrical activity, the catalytic activity of certain enzymes, the stability and activity of DNA and RNA, oxido-reduction reactions for energy supply, and protection against oxidative damage. Calcium and strontium are the only cations to play a significant role in the hard structures of living organisms. Strontium was in fact first detected by William Cruikshank in 1787 in the mineral strontianite (SrCO3), originally found near the Scottish village of Strontian in Argyle. But it was Davy who first isolated strontium in 1808, using the same method he had used to isolate calcium. Others metals, such as lead and mercury, can accumulate in living organisms, but are either inactive or toxic, rather than having a specialised function as such. Interestingly, aluminium has no known biological role in any living system. Yet aluminium is the most common metallic element in the Earth's crust, making up nearly 40% of the metallic elements. But aluminium is very reactive and in fact is quite toxic. So why has Natural Selection never exploited this? Perhaps this is the most compelling evidence that life really did begin outside our planet, somewhere in space – the panspermia hypothesis (Hoyle and Wickramasinghe, 2000).

Table 1.3 The elements of life.

Note: Boron is a metalloid, showing both metallic and non-metallic properties.

Non-metals, on the other hand, are essential for all living structures – hard and soft. These include the hard structures of bone, teeth and shells, and the soft structures of cell walls, membranes, microtubules and microfilaments invisible to the naked eye. Hydrogen, carbon, nitrogen, oxygen and sulphur are, of course, the elements that make up all proteins. But some non-metals have other specific functions, such as the unique role of oxygen or sulphur in energy supply through oxido-reduction reactions, and the oxyhalides OCl–, OBr– and OSCN– produced by phagocytes in order to kill invading microorganisms or by some fertilised eggs to prevent another sperm fusing. Non-metals make up several important gases in living organisms including O2, CO2, NO, H2, H2S, CH4 and C2H4. As anions (negatively charged ions) non-metals contribute to the osmotic, electrical and acidic balance with the cations inside and outside cells. And we must not forget water – 70% of the net weight of most living systems is H2O. In fact you could argue that all life has evolved to do is to process water.

Nine metallic elements (sodium, potassium, calcium, magnesium, manganese, cobalt, iron, copper and zinc) and seven non metals (hydrogen, nitrogen, carbon, oxygen, phosphorous, sulphur and chlorine) are essential for virtually all living systems. The others listed in Table 1.3 have specific functions in particular cell types or organisms.

Calcium has four key roles in life:

Structural:

hard structures – through calcium precipitates;

soft structures – through binding to membranes, cell adhesion and internal granules.

Electrical – through Ca²+-dependent currents across membranes.

Catalytic cofactor – through binding to enzymes, and other proteins, outside of cells.

Universal intracellular regulator – a wide range of biological processes.

It is the last of these that is the focus of this book. Ca²+ has a unique role as a switch, causing cells, organs and even entire organisms to cross the Rubicon (Campbell, 1994). As a result a biological event occurs. A nerve fires, a muscle contracts, a heart beats, an insulin cell secretes, a luminous jellyfish flashes, an egg divides and differentiates, a plant survives cold shock or wind, a bacterium competes with others in the Babal of the gut, a cell defends itself from attack or signals itself to die. All of these processes are triggered by a tiny release of the calcium ion inside the cell.

1.4 Natural Occurrence of Calcium

1.4.1 Isotopes of Calcium

There are 24 known calcium isotopes. Six are naturally occurring (Table 1.4), 97% in Nature being ⁴⁰Ca with 20 protons and 20 neutrons. ⁴³Ca has 20 protons and 23 neutrons. It is used in nuclear magnetic resonance (NMR) spectroscopy to study solid structures and can be used to study Ca²+ binding to biological molecules such as proteins. Many radioactive isotopes of calcium have been generated (Table 1.4). Most have half-lives (t1/2) which are too short for them to occur naturally, though one radioactive isotope of calcium has a long enough half-life (10⁵ years) to be found naturally. The most useful artificial calcium isotope is ⁴⁵Ca, which has been used frequently to study calcium fluxes across biological membranes and calcium binding to macromolecules (Borle, 1990; Stephenson, 1987). ⁴¹Ca, with a half-life of 102 000 years, is found in the cosmos, produced by neutron activation of ⁴⁰Ca.

Table 1.4 Isotopes of calcium.

Data from Kaye and Layby (1959), The Handbook of Chemistry and http://en.wikipedia.org/wiki/Isotopes_of_calcium.

1.4.2 Geology of Calcium

Calcium is one of the most common elements in the Earth's crust, accounting for 3% in total (Day, 1963; Fyfe, 1974; Mason, 1991; Tegethoff, 2001). On a molar basis, aluminium is the most common metal at nearly 38%. As I have already pointed out, it is a puzzle therefore that aluminium has never been exploited by any known living organism. After aluminium, in abundance, comes sodium at 14% of the metallic elements. Calcium is almost as abundant, at 13.6% (Figure 1.2). Of the non-metals, oxygen is by far the most abundant at 71.4%. Oxygen is the most important biological ligand for calcium when it binds to proteins, other macromolecules and small molecules. Over 700 calcium-containing minerals are known. These occur in all three main types of rock: igneous, metamorphic and sedimentary (Table 1.5), many forming