Dataset Information

Light-Regulated Transcription of a Mitochondrial-Targeted K⁺ Channel.

ABSTRACT: 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.

SUBMITTER: Engel AJ

PROVIDER: S-EPMC7699372 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Publications

Light-Regulated Transcription of a Mitochondrial-Targeted K+ Channel.

Engel Anja J AJ Winterstein Laura-Marie LM Kithil Marina M Langhans Markus M Moroni Anna A Thiel Gerhard G

Cells 20201119 11

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 cryptochro ...[more]

PMID: 33228123

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Project description:Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2-3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.

Dataset Information

Light-Regulated Transcription of a Mitochondrial-Targeted K⁺ Channel.

Publications

Light-Regulated Transcription of a Mitochondrial-Targeted K<sup>+</sup> Channel.

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