Seeing is believing! Imaging Ca2+-signalling events in living cells

Abstract

Ever since it was shown that maintenance of muscle contraction required the presence of extracellular Ca2+, evidence has accumulated that Ca2+ plays a crucial role in excitation–contraction coupling. This culminated in the use of the photoprotein aequorin to demonstrate that [Ca2+]i increased after depolarization but before contraction in barnacle muscle. Green fluorescent protein was extracted from the same jellyfish as aequorin, so this work also has important historical links to the use of fluorescent proteins as markers in living cells. The subsequent development of cell-permeant Ca2+ indicators resulted in a dramatic increase in related research, revealing Ca2+ to be a ubiquitous cell signal. High-speed, confocal Ca2+ imaging has now revealed subcellular detail not previously apparent, with the identification of Ca2+ sparks. These act as building blocks for larger transients during excitation–contraction coupling in cardiac muscle, but their function in smooth muscle appears more diverse, with evidence suggesting both ‘excitatory’ and ‘inhibitory’ roles. Sparks can activate Ca2+-sensitive Cl− and K+ currents, which exert positive and negative feedback, respectively, on global Ca2+ signalling, through changes in membrane potential and activation of voltage-operated Ca2+ channels. Calcium imaging has also demonstrated that agonists that appear to evoke relatively tonic increases in average [Ca2+]i at the whole tissue level often stimulate much higher frequency phasic Ca2+ oscillations at the cellular level. These findings may require re-evaluation of some of our models of Ca2+ signalling to account for newly revealed cellular and subcellular detail. Future research in the field is likely to make increasing use of genetically coded Ca2+ indicators expressed in an organelle- or tissue-specific manner.