Excitation-contraction coupling in muscle cells is initiated by a restricted membrane depolarization delimited within the neuromuscular junction. This targeted depolarization triggers an action potential that propagates and induces a global cellular calcium response and a consequent contraction. To date, numerous studies have investigated this excitation-calcium response coupling by using different techniques to depolarize muscle cells. However, none of these techniques mimic the temporal and spatial resolution of membrane depolarization observed in the neuromuscular junction. By using optogenetics in C2C12 muscle cells, we developed a technique to study the calcium response following membrane depolarization induced by photostimulations of membrane surface similar or narrower than the neuromuscular junction area. These stimulations coupled to confocal calcium imaging generate a global cellular calcium response that is the consequence of a membrane depolarization propagation. In this context, this technique provides an interesting, contactless and relatively easy way of investigation of calcium increase/release as well as calcium decrease/re-uptake triggered by a propagated membrane depolarization.

Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on channels, such as transient receptor potential (TRP) channels, to be constitutively opened and strongly depend on membrane potential and a calcium driving force. We developed an optogenetic approach based on the expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using C2C12 cells, we found that halorhodopsin can be used to achieve a finely tuned control of membrane polarization. Escalating the membrane polarization by incremental changes in light led to a concomitant increase in CCE through transient receptor potential vanilloid 2 (TRPV2) channels. Moreover, light-induced calcium entry through TRPV2 channels promoted cell migration. Our study shows for the first time that by modulating CCE and related physiological responses, such as cell motility, halorhodopsin serves as a potentially powerful tool that could open new avenues for the study of CCE and associated cellular behaviors.

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25Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell 26 dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on 27 channels, such as TRP, to be constitutively opened and strongly depend on membrane 28 potential and calcium driving force. We developed an optogenetic approach based on 29 expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using 30 C2C12 cells, we found that halorhodopsin can be used to achieve a finely-tuned control of 31 membrane polarization. Escalating the membrane polarization by incremental changes in 32 light led to a concomitant increase in CCE through TRPV2 channels. Moreover, light-33 induced calcium entry through TRPV2 channels promoted cell migration. Our study shows 34 for the first time that by modulating CCE and related physiological responses such as cell 35 motility, halorhodopsin serves as a potentially powerful tool that could open new avenues 36 for the study of CCE and associated cellular behaviors.37 38

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