The inner membranes of mitochondria contain several types of K⁺ channels, which modulate the membrane potential of the organelle and contribute in this way to cytoprotection and the regulation of cell death. To better study the causal relationship between K⁺ channel activity and physiological changes, we developed an optogenetic platform for a light-triggered modulation of K⁺ conductance in mitochondria. By using the light-sensitive interaction between cryptochrome 2 and the regulatory protein CIB1, we can trigger the transcription of a small and highly selective K⁺ channel, which is in mammalian cells targeted into the inner membrane of mitochondria. After exposing cells to very low intensities (≤0.16 mW/mm²) of blue light, the channel protein is detectable as an accumulation of its green fluorescent protein (GFP) tag in the mitochondria less than 1 h after stimulation. This system allows for an in vivo monitoring of crucial physiological parameters of mitochondria, showing that the presence of an active K⁺ channel causes a substantial depolarization compatible with the effect of an uncoupler. Elevated K⁺ conductance also results in a decrease in the Ca²⁺ concentration in the mitochondria but has no impact on apoptosis.

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