1054 J. G. McGeown Exp Physiol 95.11 pp 1049–1060 in which the membrane potential of a smooth muscle cell could be clamped at a speciﬁc voltage and transmembrane ion currents recorded, had revealed brief, relatively short- lived and outwardly directly K+ currents (Fig. 6A). These were given the descriptive title ‘spontaneous transient outward currents (STOCs)’ (Benham & Bolton, 1986). When caffeine was applied to release stored Ca2+ by activating RyRs, there was an initial burst of STOC activity, followed by relative quiescence. This response could be inhibited by introducing EGTA, a Ca2+ chelator, to the intracellular solution via the recording pipette, indicating that the current was activated by a rise in [Ca2+]i. Taken together, these ﬁndings suggested that STOCs reﬂected spontaneous Ca2+ release from the sarcoplasmic reticulum via RyRs, leading to the activation of Ca2+-sensitive K+ channels conducting the outward current (I KCa). Such channels (also known as large-conductance K+ channels, or BK channels) had already been identiﬁed in smooth muscle (Walsh & Singer, 1981; Benham et al. 1986). It was not until 1995, however, that this indirect evidence for localized spontaneous Ca2+ release in smooth muscle was conﬁrmed by the direct visualization of Ca2+ sparks, again using confocal imaging of Ca2+ indicator ﬂuorescence (Fig. 6B; Nelson et al. 1995). Both sparks and STOCs were inhibited by using ryanodine to block RyR activity and by agents that depleted the Ca2+ stores. Calcium imaging therefore allowed spontaneous Ca2+ release to be directly observed in smooth muscle cells, conﬁrming a hypothesis originally postulated on the basis of indirect evidence. What do sparks do? Physiology is driven by functional questions. Usually, this expresses itself in the desire to understand the mechanisms responsible for the function of a molecule, cell, organ or organism. In the case of Ca2+ sparks, however, we are presented with a cell mechanism whose function needs to be deﬁned. In the case of cardiac muscle, it was immediately proposed that they might act as fundamental building blocks from which larger and global Ca2+ transients are constructed during the excitation–contraction process (Cheng et al. 1993). This view has been conﬁrmed over the intervening years. Depolarization of cardiac myocytes activates voltage- operated (L-type) Ca2+ channels in the plasma membrane, leading to an inﬂux down the electrochemical gradient from the extracellular ﬂuid. This stimulates the opening of near-membrane RyRs, which are directly sensitive to the local [Ca2+]i, leading to an almost synchronous cell-wide Ca2+ rise and contraction (Cheng & Lederer, 2008). Individual sparks are not easily identiﬁed in confocal linescans of normal Ca2+ transients but become obvious when relatively small depolarizations are applied. Thus, in cardiac muscle at least, sparks act as excitatory events whose summation generates the intracellular signal responsible for contraction. The story in smooth muscle, however, does not appear to be so straightforward. In the initial report of Ca2+ sparks in arterial smooth muscle, considerable evidence Figure 5. Application of confocal microscopy to Ca2+ imaging in living cells Cells are loaded with a ﬂuorescent Ca2+ indicator (e.g. ﬂuo-4) and excited with a scanning laser. A, in XY scanning mode a single image plane is scanned repeatedly, allowing any [Ca2+]i rises to be recorded and their location identiﬁed. B, line scanning greatly increases the speed of acquisition by imaging only along a single line rather than a full XY plane. The resulting images show how [Ca2+]i changes locally at each point on the line as time passes. This is particularly useful when imaging fast events, such as Ca2+ sparks. C 2010 The Author. Journal compilation C 2010 The Physiological Society