1052 J. G. McGeown Exp Physiol 95.11 pp 1049–1060 be studied. The only alternative approach, which involved impaling cells with a Ca2+-selective microelectrode, was equally laborious and technically difﬁcult, and could not always follow very rapid changes in [Ca2+]i (Lee, 1981). Better, more user-friendly Ca2+ indicators were clearly needed. This challenge was identiﬁed and taken on by Roger Tsien, ﬁrst in the University of Cambridge and later in the University of California, Berkeley. He recognized that, just as physiologists had been obliged to design and manufacture the electronic equipment needed for the study of neuronal action potentials in a previous generation, so too there was a need to develop the new chemical tools needed for cell-signalling research. A range of ﬂuorescent, Ca2+-sensitive dyes resulted from this work, along with a chemical strategy allowing them to be easily introduced into intact cells (Kresge et al. 2006). Acetoxymethyl esters are lipid soluble and cell permeant but are readily hydrolysed by intracellular enzymes, releasing the active dye in its anionic form. This remains trapped within the cell, and changes in ﬂuorescence can then be used as a measure of [Ca2+]i. Based on its use in research papers, the most successful of these compounds was fura-2, a ratiometric Ca2+ indicator. These dyes circumvent movement artefacts and open the door to calibration of the signal in terms of absolute [Ca2+] (Grynkiewicz et al. 1985). As is often the case, the availability of new research tools that were relatively easily applied led to a massive explosion in Ca2+-signalling research, with many thousands of papers published in the last 25 years. It quickly became apparent that increases in [Ca2+]i were involved in the transduction of external stimuli into cellular responses in almost every cell type studied. Calcium signalling is genuinely ubiquitous, affecting a wide variety of molecular targets in many different types of cells. No one reference can really capture this diversity, but a simple PubMed search for the term ‘Ca-signalling’ generated over 7900 hits in June 2010. The importance of Ca2+ signalling is indicated by the very low basal [Ca2+]I found in most cells, which is maintained by a variety of Ca2+ pumps and exchangers. This allows relatively small concentration increases to be resolved by Ca2+- sensitive proteins. An analogy can be found in astronomy, in which viewing conditions improve as background ‘light pollution’ decreases, making faint objects easier to detect. At about 100 nM or so, resting [Ca2+]i is more than four orders of magnitude lower than the free extracellular [Ca2+]i in mammals, and at least 100 times less than that in the sarcoplasmic/endoplasmic reticulum, the major intracellular Ca2+ store (McCarron et al. 1992; Wray & Burdyga, 2010). Despite the large electrochemical gradients involved, however, Ca2+ does not simply ﬂood into the cytosol, since it cannot readily cross lipid membranes. Calcium can only diffuse into the cell if appropriate ion channels are activated, providing a Ca2+-permeable access route. Many such channels have been identiﬁed in the cell membranes of both excitable and non-excitable tissues, and are activated by very diverse stimuli. Intracellular stores can release Ca2+, mainly via opening of ryanodine receptor (RyR)-gated or inositol 1,4,5-trisphosphate receptor (IP3R)-gated release channels. Some of these Ca2+ channels will be mentioned Figure 4. A direct demonstration that Ca2+ is involved in excitation–contraction coupling Barnacle muscle ﬁbres were injected with aequorin, a protein which chemiluminesces when it binds Ca2+. The ﬁbres were stimulated electrically, mimicking depolarization by an action potential, and changes in membrane potential, [Ca2+] (light generated by aequorin) and tension were measured simultaneously. Depolarization, a rise in [Ca2+]i and contraction occurred in that sequence, consistent with the hypothesis that Ca2+ signalling plays an important role in excitation–contraction coupling. Reproduced with permission from Ashley & Ridgway (1970). C 2010 The Author. Journal compilation C 2010 The Physiological Society