Optogenetic control of mitochondrial metabolism and Ca ²⁺ signaling by mitochondria-targeted opsins

Significance

Mitochondrial functions depend on the steep H ⁺ electrochemical gradient (ΔμH ⁺) across their inner membrane. The available tools for controlling this gradient are essentially limited to inhibitors of the respiratory chain or of the H ⁺ ATPase or to uncouplers, poisons plagued by important side effects and that lack both cell and spatial specificity. We show here that, by transfecting cells with the cDNA encoding channelrhodopsins specifically targeted to the inner mitochondrial membrane, we can obtain an accurate and spatially confined, light-dependent control of mitochondrial membrane potential and, as a consequence, of a series of mitochondrial activities ranging from electron transport to ATP synthesis and Ca ²⁺ signaling.

Abstract

Key mitochondrial functions such as ATP production, Ca ²⁺ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH ⁺) across the inner membrane. Although several drugs can modulate ΔμH ⁺, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψ _m) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca ²⁺ dynamics, and respiratory metabolism. By directly modulating Δψ _m, the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.