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.