Our laboratory recently discovered that blue light photoactivation of insect Cryptochrome (Cry) cause rapid membrane depolarization and up to 300% increased action potential firing rate over baseline dark spontaneous firing in central brain neurons (Sheeba et al., 2007; Fogle et al., 2011). The electrophysiological light response is robust in the absence of all opsin-based classical photoreceptor inputs (Fogle et al., 2011). Genetically targeted expression of Cry in normally light-insensitive olfactory neurons confers electrophysiological light responsiveness, indicating that Cry expression may be used for optogenetic applications (Fogle et al., 2011). A combination of molecular-genetic and pharmacological experiments indicate that Cry's light sensitivity is mediated through light-activated changes in the redox state of the flavin adenine dinucleotide (FAD) chromophore bound to dCry which then couples to a redox sensor in cytoplasmic potassium channel subunits and modulate potassium channel activity. We propose to extend these findings by determining the precise molecular mechanism of how light activated Cry undergoes an intramolecular transfer of redox state from the flavin chromophore to the protein surface of Cry by testing mutants which lack a well conserved tri-tryptophan motif characterized in other Cry proteins as conducting redox signals. We will then test the hypothesis that redox transfer takes place to target proteins in the membrane. Based on strong preliminary data that membrane coupling of Cry's light activated redox state occurs through voltage gated potassium channels, we will test the hypothesis that dCry then interacts with membrane redox-sensitive effector Hyperkinetic beta subunit (Hk) of voltage-gated potassium (Kv) channels. Our preliminary data indicates that light activation of Cry rapidly modulates cellular potassium currents and depolarizes the membrane potential. We have begun testing this hypothesis and find that the lLNv electrophysiological light response in almost completely abolished in Hk null mutant flies, suggesting that Hk is the primary membrane target for the novel dCry-based phototransduction mechanism. We will determine whether rapid translocation of dCry to the neuronal membrane increases the speed and the amplitude of the electrophysiological light response, as tested using a chemical biology-based inducible strategy. These experiments provide a unique opportunity to unravel a novel non-opsin phototransduction mechanism based on redox sensing. We have also the first opportunity to examine real-time actions of Cry in vivo and the possibility of determining a biological function for the highly conserved redox sensor in KvBeta subunits. As Cry's chromophore, FAD, is the ubiquitously expressed, our work may provide the basis of a new Vitamin B-based optogenetic technology applicable to cells that do not synthesize adequate levels of retinal.

Public Health Relevance

We propose to examine the molecular mechanism that couples blue light activated Cryptochrome to rapid membrane depolarization and increased action potential firing rate. Our preliminary data shows that this novel phototransduction mechanism is based on a fast acting redox couple between light activated Cryptochrome and membrane ion channel subunits that act as redox sensors. As Cryptochrome uses a ubiquitous flavin chromophore for light activation, the Cryptochrome-to-membrane electrical signaling couple may be developed further for alternative non-opsin based optogenetic technologies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM102965-03
Application #
8915215
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Barski, Oleg
Project Start
2013-08-01
Project End
2017-05-31
Budget Start
2015-06-01
Budget End
2016-05-31
Support Year
3
Fiscal Year
2015
Total Cost
$282,017
Indirect Cost
$92,017
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Baik, Lisa Soyeon; Recinos, Yocelyn; Chevez, Joshua A et al. (2018) Circadian modulation of light-evoked avoidance/attraction behavior in Drosophila. PLoS One 13:e0201927
Sun, Yanjun; Nitz, Douglas A; Holmes, Todd C et al. (2018) Opposing and Complementary Topographic Connectivity Gradients Revealed by Quantitative Analysis of Canonical and Noncanonical Hippocampal CA1 Inputs. eNeuro 5:
Grieco, Steven F; Holmes, Todd C; Xu, Xiangmin (2018) Neuregulin directed molecular mechanisms of visual cortical plasticity. J Comp Neurol :
Baik, Lisa S; Fogle, Keri J; Roberts, Logan et al. (2017) CRYPTOCHROME mediates behavioral executive choice in response to UV light. Proc Natl Acad Sci U S A 114:776-781
Ni, Jinfei D; Baik, Lisa S; Holmes, Todd C et al. (2017) A rhodopsin in the brain functions in circadian photoentrainment in Drosophila. Nature 545:340-344
Sun, Yanjun; Grieco, Steven F; Holmes, Todd C et al. (2017) Local and Long-Range Circuit Connections to Hilar Mossy Cells in the Dentate Gyrus. eNeuro 4:
Das, Antara; Holmes, Todd C; Sheeba, Vasu (2016) dTRPA1 in Non-circadian Neurons Modulates Temperature-dependent Rhythmic Activity in Drosophila melanogaster. J Biol Rhythms 31:272-88
Sun, Yanjun; Ikrar, Taruna; Davis, Melissa F et al. (2016) Neuregulin-1/ErbB4 Signaling Regulates Visual Cortical Plasticity. Neuron 92:160-173
Xu, Xiangmin; Ikrar, Taruna; Sun, Yanjun et al. (2016) High-resolution and cell-type-specific photostimulation mapping shows weak excitatory vs. strong inhibitory inputs in the bed nucleus of the stria terminalis. J Neurophysiol 115:3204-16
Xu, Xiangmin; Sun, Yanjun; Holmes, Todd C et al. (2016) Noncanonical connections between the subiculum and hippocampal CA1. J Comp Neurol 524:3666-3673

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