Two-photon activation of endogenous store-operated calcium channels without optogenetics

Store-operated calcium (SOC) channels, regulated by intracellular Ca²⁺ store, are the essential pathway of calcium signaling and participate in a wide variety of cellular activities such as gene expression, secretion and immune response¹. However, our understanding and regulation of SOC channels are mainly based on pharmacological methods. Considering the unique advantages of optical control, optogenetic control of SOC channels has been developed². However, the process of genetic engineering to express exogenous light-sensitive protein is complicated, which arouses concerns about ethic difficulties in some research of animal and applications in human. In this report, we demonstrate rapid, robust and reproducible two-photon activation of endogenous SOC channels by femtosecond laser without optogenetics. We present that the short-duration two-photon scanning on subcellular microregion induces slow Ca²⁺ influx from extracellular medium, which can be eliminated by removing extracellular Ca2+. Block of SOC channels using various pharmacological inhibitors or knockdown of SOC channels by RNA interference reduce the probability of two-photon activated Ca²⁺ influx. On the contrary, overexpression of SOC channels can increase the probability of Ca²⁺ influx by two-photon scanning. These results collectively indicate Ca²⁺ influx through two-photon activated SOC channels. Different from classical pathway of SOC entry activated by Ca²⁺ store depletion, STIM1, the sensor protein of Ca²⁺ level in endoplasmic reticulum, does not show any aggregation or migration in this two-photon activated Ca²⁺ influx, which rules out the possibility of intracellular Ca²⁺ store depletion. Thereby, we propose this all-optical method of two-photon activation of SOC channels is of great potential to be widely applied in the research of cell calcium signaling and related biological research.