Optogenetic regulation of Ca 2+ signaling April 15, 2013 Class objectives: What is optogenetics? Mammalian rhodopsin. Biophysics of channelrhodopsin-2. Opto-XR Optically-induced dimerization

“Optogenetics is the combination of optical and molecular strategies to monitor and control designated molecular and cellular activities in living tissues and cells using genetically encoded photosensitive proteins.” What is optogenetics?

Mammalian rhodopsin 11-cis-retinal

11-cis- retinylidene All-trans- retinylidene

Channelrhodopsin-2 is a light- activated cation channel Green alga Chlamydomonas reinhardtii Nagel G et al. PNAS 2003;100:

Action spectra of channelrhodopsins

Prigge M et al. J. Biol. Chem. 2012;287: ChR chimeras with different spectral properties

Prigge M et al. J. Biol. Chem. 2012;287: Dual light excitation and ion selectivity for blue and green light-absorbing ChRs.

RD Airan et al. Nature (2009) Signalling specificity and in vivo functionality of Opto-XRs

RD Airan et al. Nature 000, 1-5 (2009) doi: /nature07926 OptoXR: optogenetic control of intracellular signal transduction.

Opto-  1AR activation of TRPV4 channels

Activation wavelength (nm) ReversibleReference Flavin-binding, kelch repeat, F box 1 (FKF1) and gigantea (GI) Blue lightYes (hours)Yazawa et al Phytocrome B and PIF6 Associates with red light and dissociates with far-red light Yes (seconds) Liu et al CIB1 and cryptochrome 2 (CRY2), a blue light– absorbing photosensor that binds CIB1 in its photoexcited state9 Blue lightYes (minutes) Kennedy et al Systems for optically-induced protein interactions

Light-induced translocation of PKC  FKF1-GI system

Idevall-Hagren O et al. PNAS 2012;109:E2316-E2323 Rapid light-induced recruitment of a 5-ptase to the plasma membrane

Idevall-Hagren O et al. PNAS 2012;109:E2316-E2323 Blue light-induced recruitment of a 5- phosphatase to the plasma membrane rapidly decreases KCNQ2/3 current.

Intermission

Jia et al.(2011) Fig. 1

Jia et al.(2011) Fig. 2a-b

Jia et al.(2011) Fig. 2c-e

Jia et al.(2011) Fig. 3a-c

Jia et al.(2011) Fig. 3d-f

Jia et al.(2011) Fig. 4a-b

Jia et al.(2011) Fig. 3c-g

Jia et al.(2011) Supplemental Fig. 1a-b

Jia et al.(2011) Supplemental Fig. 1c-d

Supplemental movies Jia et al.(2011) Supplemental Movies