What's in store for Ca²⁺ oscillations?

At fertilization, at every heartbeat, and during a myriad of other Ca²⁺-regulated cellular responses, Ca²⁺ signals are delivered to the cell as Ca²⁺ oscillations. Such digital signals, analogous to action potentials in neurones, are robust and economical, they may protect cells from the toxic effects of sustained increases in cytosolic [Ca²⁺], and they allow information to be encoded in the frequency, amplitude and duration of the Ca²⁺ spikes (Berridge et al. 2000). Certainly there is evidence that cellular events may be tuned to respond optimally to specific frequencies of Ca²⁺ spiking (Berridge et al. 2000). The mechanisms underlying Ca²⁺ oscillations are diverse, and while all include elements of negative feedback by Ca²⁺, they may be driven entirely by events at the plasma membrane, entirely by release of Ca²⁺ from intracellular stores, or by an interplay between intra- and extra-cellular sources of Ca²⁺. In heart, for example, a slowly depolarizing membrane potential triggers opening of voltage-gated Ca²⁺ channels, Ca²⁺ enters, triggering further Ca²⁺ release from the sarcoplasmic reticulum, and the increase in sarcoplasmic [Ca²⁺] then both initiates contraction and promotes repolarization. In other cells, Ca²⁺ oscillations are driven entirely by interplay between voltage-gated Ca²⁺ channels and Ca²⁺-sensitive channels that regulate membrane potential. The mechanisms controlling the Ca²⁺ oscillations evoked by receptors that stimulate IP₃ formation are less clear. In some cells, levels of IP₃ may themselves oscillate and drive periodic release of Ca²⁺ from intracellular stores (Nash et al. 2001), but in other cells Ca²⁺ oscillations occur when IP₃ levels are probably stable. The latter are thought to result from episodic release of intracellular Ca²⁺ stores driven by the co-regulation of IP₃ receptors by Ca²⁺ and IP₃. But what role has Ca²⁺ entry to play in such Ca²⁺ oscillations? Ca²⁺ entry is certainly required to sustain these Ca²⁺ oscillations, but does it serve only to replenish stores or does it play a more central role in the oscillatory mechanism, and are all Ca²⁺ entry pathways equally effective in supporting Ca²⁺ oscillations?

Both questions are addressed in a paper (Bird & Putney, 2005) from this issue of The Journal of Physiology. Gd³⁺ is often used to inhibit Ca²⁺ channels, indeed at low concentrations (∼1 μm) it can be used selectively to inhibit store-operated Ca²⁺ (SOC) entry, but at higher concentrations it also inhibits plasma membrane Ca²⁺ pumps. The authors cleverly exploit the ability of high concentrations of Gd³⁺ to inhibit both Ca²⁺ entry and extrusion effectively to insulate HEK-293 cells from Ca²⁺ exchanges with the environment. Under these conditions, activation of endogenous muscarinic acetylcholine receptors evokes Ca²⁺ oscillations that are sustained, and in keeping with earlier work, the frequency rather than the amplitude of the individual spikes increases with stimulus intensity. The oscillatory mechanism does not therefore require Ca²⁺ entry, that is required only to replenish Ca²⁺ lost from the stores as cells restore the cytosolic [Ca²⁺] to its resting level between spikes (Sneyd et al. 2004). It is noteworthy that there is no detectable reduction in the overall Ca²⁺ content of the intracellular stores as the Ca²⁺ oscillations fail, suggesting either that only a small subset of stores contributes to Ca²⁺ spiking or that the underlying mechanism depends critically on their complete refilling. The latter would be consistent with various dynamic models of Ca²⁺ oscillations (Sneyd et al. 2004).

Under normal conditions, where plasma membrane Ca²⁺ pumps actively buffer cytosolic [Ca²⁺], Ca²⁺ entry is important, but the route through which Ca²⁺ enters HEK cells to sustain oscillations is contentious. Shuttleworth argues that at the low stimulus intensities that evoke Ca²⁺ oscillations, all Ca²⁺ entry is via an arachidonic acid-activated Ca²⁺ channel (I_ARC) and that this is exclusively responsible for sustaining Ca²⁺ oscillations (Shuttleworth, 2004). Bird and Putney conclude that because oscillations are blocked by Gd³⁺ (1 μm) and 2-APB, they are sustained entirely by Ca²⁺ entering through the ubiquitously expressed SOC pathway (Bird & Putney, 2005). How can two labs reach such contradictory conclusions? A partial answer comes from the final experiments in the Bird and Putney paper, where they transfect cells with TRPC3 to create an additional non-SOC and Gd³⁺-insensitive Ca²⁺ entry pathway. In these cells, the new Ca²⁺ entry pathway also supports Ca²⁺ oscillations. There seems not therefore to be any preferential relationship between specific Ca²⁺ entry pathways and their ability to support Ca²⁺ oscillations by fuelling store refilling: SOC, TRPC3 and I_ARC are all effective. This is perhaps surprising because the intimate relationship between store depletion and activation of SOC led many to suppose that SOC might preferentially couple to store refilling. The disparity between the two labs thus boils down to understanding why each apportions responsibility for Ca²⁺ entry to a different pathway, rather than to any fundamental difference in the mechanism driving Ca²⁺ oscillations.