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 (IARC) 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 IARC 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.
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Jan 9, 2024
Chang-Rok Choi + 8
Open Access
