Exp Physiol 95.11 pp 1049–1060 Ca2+ events in living cells 1051 Since these early observations, a large amount of evidence has accumulated suggesting that changes in intracellular [Ca2+] ([Ca2+]i) play a crucial role in the control of cell function, particularly in muscle. The sliding ﬁlament hypothesis of striated muscle contraction was established independently (by two different Huxleys) based on structural and functional experiments in the 1950s (see review, Huxley, 2000). By the early 1960s it was apparent that biochemical extracts of the contractile machinery could interact in the test tube to form a superprecipitate. This process required millimolar MgATP as an energy source but was also highly sensitive to [Ca2+], with measurable superprecipitation and ATP breakdown when [Ca2+] exceeded 10−7 M (Weber & Winicur, 1961). This mimicking of muscle contraction in a test tube suggested that Ca2+ played some sort of role in controlling the contractile process. The development of techniques allowing muscle cells to be permeabilized, so that intracellular and extracellular [Ca2+] become equal, demonstrated a very similar relationship between [Ca2+]i and contractile force in permeabilized ﬁbres and the Ca2+ dependence of superprecipitation, with activation of muscle contraction when [Ca2+]i exceeds 100 nM and maximal contraction above 1 μM (Fig. 3; Filo et al. 1965). Interestingly, the same relationship applied to both smooth and striated muscle, even though we now know there are major differences in the way contraction is regulated in these different muscle types (Fig. 3). We will return to this point later. The evidence up to this point supported a model in which excitation of a striated muscle ﬁbre by the propagation of an action potential across the cell membrane was coupled to mechanical contraction via the intracellular machinery through a Ca2+-dependent signalling process. However, this evidence was largely indirect and so the hunt was now on for some way to record changes in [Ca2+]i during electrically evoked muscle contraction in intact cells, so as to more closely reﬂect physiological events. Measurements of [Ca2+]i in living cells The relevant information was eventually derived from experiments using a Ca2+ indicator from a most unlikely source. Certain species of luminescent jellyﬁsh manufacture the photoprotein aequorin, which emits a blue glow when it interacts with Ca2+. The protein was ﬁrst isolated and puriﬁed from Aequoria aequoria by Osamu Shimomura, a biochemist with a keen interest in light-emitting molecules from biological sources (bioluminescence), while working in the laboratory of Frank Johnson at Princeton. They showed that the amount of light released could be used as a measure of the [Ca2+] in an aequorin-containing solution, opening the way C 2010 The Author. Journal compilation C 2010 The Physiological Society for biological applications of the technique (Shimomura et al. 1963). Aequorin was used to demonstrate that Ca2+ signalling played a key role in excitation–contraction coupling within barnacle muscle (Ashley & Ridgway, 1968, 1970). This has large-diameter ﬁbres, greatly facilitating intracellular aequorin injection. Contractions were stimulated by electrically depolarizing the ﬁbres, and the resulting changes in membrane potential, Ca2+- dependent light emission by the aequorin and contractile force measured simultaneously (Fig. 4). This allowed both the amplitudes and the temporal sequencing of these events to be assessed. Membrane depolarization was followed by a rise in [Ca2+], which was, in turn, followed by muscle contraction. Since cause must precede effect, this ruled out the possibility that the [Ca2+]i increase was some sort of epiphenomenon or ‘side- effect’ of contraction itself, but was consistent with the hypothesis that excitation of the muscle ﬁbre causes an increase in intracellular [Ca2+], which activates the formation of cross-bridges between myosin and actin. We now know that, in striated muscle, this results from the binding of Ca2+ to the troponin–tropomyosin complex on the thick myoﬁlament, thereby relieving steric inhibition of cross-bridge formation between myosin heads and actin (Galinska-Rakoczy et al. 2008). Calcium also plays an important role in smooth muscle signalling using very different downstream mechanisms, with Ca2+– calmodulin activating myosin light chain kinase. The resulting phosphorylation upregulates myosin ATPase activity, releasing the chemical energy needed for cross- bridge cycling and cell shortening (Itoh et al. 1989). This work established the involvement of intracellular Ca2+ in excitation–contraction coupling. Aequorin was difﬁcult to use, however, since it had to be isolated and puriﬁed from jellyﬁsh and then injected into each cell to Figure 3. Effect of changing [Ca2+] on contraction in permeabilized muscle Permeabilized muscle ﬁbre preparations were used, allowing intracellular [Ca2+] to be controlled simply by changing the extracellular [Ca2+]. The otherwise intact ﬁbres developed active tension when [Ca2+] exceeded 100 nM, with maximal contraction above 1 μM. Interestingly, the relationships in striated and smooth muscles were almost identical. Reproduced with permission from Filo et al. (1965).