Martin D. Bootman

The versatility and universality of calcium signalling

The universality of calcium as an intracellular messenger depends on its enormous versatility. Cells have a calcium signalling toolkit with many components that can be mixed and matched to create a wide range of spatial and temporal signals. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of calcium being highly toxic. Exceeding its normal spatial and temporal boundaries can result in cell death through both necrosis and apoptosis.

Calcium signalling: dynamics, homeostasis and remodelling

Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.

Calcium - a life and death signal

One of the most versatile and universal signalling agents in the human body is the calcium ion, Ca2+. How does this simple ion act during cell birth, life and death, and how does it regulate so many different cellular processes?

2-Aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release

Since its introduction to Ca2+ signaling in 1997, 2‐aminoethoxydiphenyl borate (2‐APB) has been used in many studies to probe for the involvement of inositol 1,4,5‐trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2‐APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2‐APB has thrown up some interesting results, particularly with respect to store‐operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2‐APB can be useful in probing certain aspects of Ca2+ signaling.—Bootman, M. D., Collins, T. J., Mackenzie, L., Roderick, H. L., Berridge, M. J., Peppiatt, C. M. 2‐Aminoethoxydiphenyl borate (2‐APB) is a reliable blocker of store‐operated Ca2+ entry but an inconsistent inhibitor of InsP3‐induced Ca2+ release. FASEB J. 16, 1145–1150 (2002)

Mitochondria are morphologically and functionally heterogeneous within cells

We investigated whether mitochondria represent morphologically continuous and functionally homogenous entities within single intact cells. Physical continuity of mitochondria was determined by three‐dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or calcein. The mitochondria of HeLa, PAEC, COS‐7, HUVEC, hepatocytes, cortical astrocytes and neuronal cells all displayed heterogeneous distributions and were of varying sizes. There was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could be seen clearly. Using fluorescence‐recovery after photobleaching, we observed that DsRed1 and calcein were highly mobile within the matrix of individual mitochondria, and that mitochondria within a cell were not lumenally continuous. Mitochondria were not electrically coupled, since only individual mitochondria were observed to depolarize following irradiation of TMRE‐loaded cells. Functional heterogeneity of mitochondria in single cells was observed with respect to membrane potential, sequestration of hormonally evoked cytosolic calcium signals and timing of permeability transition pore opening in response to tert‐butyl hydroperoxide. Our data indicate that mitochondria within individual cells are morphologically heterogeneous and unconnected, allowing them to have distinct functional properties.

The elemental principles of calcium signaling

Complexity of Intracellular Ca2+ Signals Ca2+ is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes, including secretion, contraction, and cell proliferation (Berridge, 1993; Clapham, 1995). In resting cells, the intracellular Ca2+ concentration ([Ca2+]i) is maintained at approximately 10-100 nM, and during stimulation the average [Ca2+]i can rise up to several micromolar, depending on the cell type. Such Ca2+ signals have acomplex temporal and spatial arrangement, although the mechanisms underlying the complexity are not entirely clear. Tonic [Ca2+]i, increases do not occur in many cells. Instead, Ca2+ is frequently presented to the cytoplasm in a pulsatile manner, such as the repetitive Ca2+ spikes that drive the beating heart, the rapid subplasmalemmal [Ca2+]i increases that occur during depolarization of excitable cells, and the regular Ca2+ spikes (or oscillations) observed during hormonal stimulation of many nonexcitable cell types. The spatial correlate of a Ca2+ spike is a Ca2+ wave in which Ca2+ is initially elevated in a discrete region of the cell before spreading throughout the cell as a regenerative increase. Since information is encoded in the spatiotemporal patterns of these intracellular signals, much interest is now focused on how these complex Ca2+ signals are generated.

Calcium: Calcium signalling: dynamics, homeostasis and remodelling

Ca2+ is a highly versatile intracellular signal that operates over a wide temporal range to regulate many different cellular processes. An extensive Ca2+-signalling toolkit is used to assemble signalling systems with very different spatial and temporal dynamics. Rapid highly localized Ca2+ spikes regulate fast responses, whereas slower responses are controlled by repetitive global Ca2+ transients or intracellular Ca2+ waves. Ca2+ has a direct role in controlling the expression patterns of its signalling systems that are constantly being remodelled in both health and disease.

Bcl-2 functionally interacts with inositol 1,4,5- trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.

2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels

The action of 2-aminoethoxydiphenyl borate (2-APB) on Ca(2+) signalling in HeLa cells and cardiac myocytes was investigated. Consistent with other studies, we found that superfusion of cells with 2-APB rapidly inhibited inositol 1,4,5-trisphosphate (InsP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOC). In addition to abrogating hormone-evoked Ca(2+) responses, 2-APB could antagonise Ca(2+) signals evoked by a membrane permeant InsP(3) ester. 2-APB also slowed the recovery of intracellular Ca(2+) signals consistent with an effect on Ca(2+) ATPases. The inhibitory action of 2-APB on InsP(3) receptors (InsP(3)Rs), SOC channels and Ca(2+) pumps persisted for several minutes after washout of the compound. Application of 2-APB to unstimulated cells had no effect on subsequent Ca(2+) responses suggesting that it has a use-dependent action. Mitochondria in cells treated with 2-APB showed a rapid and slowly reversible swelling. 2-APB did not cause the mitochondria to depolarise, but it reduced the extent of mitochondrial calcium uptake. Although 2-APB has been demonstrated not to affect voltage-operated Ca(2+) channels or ryanodine receptors, we found that it gave a concentration-dependent long-lasting inhibition of Ca(2+) signalling in electrically-stimulated cardiac myocytes, where InsP(3)Rs and SOC channels do not play a significant role. Our data suggest that 2-APB has multiple cellular targets, a use-dependent action, is difficult to reverse and may affect Ca(2+) signalling in cell types where InsP(3) and SOC are not active.

Calcium Signalling: More Messengers, More Channels, More Complexity

Recent studies have expanded the number of channel types and messengers that lead to Ca(2+) signals within cells. Furthermore, we are beginning to understand the complex interplay between different sources of Ca(2+).