CRAC channel activity pulsates during cytosolic Ca2+ oscillations.

Intracellular Ca2+ ions are used as second messengers throughout the phylogenetic tree. They are indispensable for diverse biological processes ranging from fertilization to cell death. In Metazoans, signaling information is conveyed via the amplitude, frequency and spatial profile of cytosolic Ca2+ oscillations. In non-excitable cells, these oscillations generally arise from regenerative release of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular stores, which are refilled by entry of Ca2+ through Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane. However, the precise contribution of these store-operated CRAC channels to Ca2+ oscillations has remained controversial for decades. One view proposes that CRAC channels remain open throughout stimulation, functioning as the pacemaker in setting Ca2+ oscillation frequency. An alternative hypothesis is that channel activity oscillates in parallel with InsP3-driven regenerative Ca2+ release. Here, by tethering a genetically encoded Ca2+ indicator to the pore- forming subunit of the CRAC channel, Orai1, we distinguish between these hypotheses and demonstrate that CRAC channel activity fluctuates in phase with cytosolic Ca2+ oscillations during physiological levels of stimulation. We also find that spatially distinct CRAC channel clusters fire in a coordinated manner, revealing that CRAC channels are not independent units but might function in a synchronized manner to provide pulses of Ca2+ signal at the same time.