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.

Codon Bias Can Determine Sorting of a Potassium Channel Protein

“…The second channel, Kesv, is sorted in a typical post-translational manner. It reaches its destination in the inner membrane of the mitochondria via the canonical TIM/TOM translocases without a mitochondrial targeting motif [ 20 , 21 ]. The decision between these two distinct trafficking pathways is made by the level of affinity of the nascent proteins for the signal recognition particle (SRP).…”

Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum

“…Expression of the eGFP‐tagged channels was performed as reported previously 18 in human embryonic kidney (HEK293) cells cultured in T25 cell culture flasks in an incubator (at 37°C and 5% CO 2 ) in standard cell culture medium [DMEM/F12‐Medium with Glutamine (Biochrom AG, Berlin, Germany) plus 10% fetal calf serum (FCS) and 1% penicillin/streptomycin].…”

“…Expression of the eGFP-tagged channels was performed as reported previously 18 in human embryonic kidney (HEK293) cells cultured in MitoTracker™ Red FM). This analysis provided three distinct sorting phenotypes in which the eGFP signal was either colocalized with (1) a mitochondria or (2) an ER-specific marker.…”

“…15 A seminal observation, which makes these proteins so interesting as a model system for protein sorting, is that one channels, Kcv, is in mammalian cells co-translationally synthesized at the translocon into the ER, 16 from where it travels via the secretory pathway to the plasma membrane. 17 In contrast the structurally similar Kesv protein is sorted post-translationally to the inner membrane of the mitochondria 18 via the canonical TIM/TOM translocases. 19 Studies with yeast have shown that a decision between the two sorting pathways occurs at the translational stage of the nascent proteins on the ribosome and is decided by their affinity for the SRP.…”