Optogenetics: A Bright Future for Voltage Gated Ion Channels - PowerPoint PPT Presentation

Optogenetics: A Bright Future for Voltage Gated Ion Channels Viviana Agus Axxam S.p.A., Milano, Italy

Presentation Summary • Hamamatsu and AXXAM: FDSS µCELL demo period Cav1.3 and ChR2 assay • Optogenetics : overview and advantages • Channelrhodopsin2 to modulate cell membrane voltage • Activation of Cav1.3 by ChR2 : recombinant assay setup • Validation of “light protocol” at FDSS µCELL : test of reference compounds • Comparison with “K + protocol” and patch -clamp data • Conclusions and future perspectives 2

FDSS μ CELL DEMO @ AXXAM Ca 2+ assay (fluorescent dyes, Fluo8 LED (blue light) luminescent photoprotein) MPdye LED (green light) • CCKAR (GPCR) • GLP1R (GPCR) • ADORA1 (GPCR) • DRD1-DRD2 (GPCR) • Enzymatic assay Glow Luminescence assay: • PPAR α , PPAR δ (NHR) • Promoter assay Genetically encoded sensor • TMEM16A (EYFP) Optogenetics Hybrid Camera • Cav1.3 (ChR2) (Fluo & Lumi) 3

Optogenetics: overview Optogenetics is a technology that combines: 1) A «genetic» component, able to target specific neuron types 2) An «optical» component, able to interact specifically with the genetic component to achieve fast control of well-defined events in specific cells of living tissue The starting point was the idea to have a system available to control the activity of specific neuron types in the brain in a better way Method of the year 2010 4

Rhodopsins: Light-gated ion channels Opsins: • Seven-transmembrane, light-responsive proteins • Rapidly translocate specific ions across the membranes of the cells in which they are expressed • Contain the Vitamin-A derived chromophore all- trans -retinal as a light capture molecule • Studied since the 1970s for their fascinating biophysical properties • Used by several different life forms that use light as energy source or sensory cue Structural simplicity, fast kinetics ► attractive tool for a rapid control of specific cellular processes , such as, for example, modulation of membrane voltage and neuronal action potentials propagation Light as activating stimulus ► more physiological , compared to other hyperpolarizing or depolarizing stimulus (for example K + injection) Possibility to target their expression to specific cell types ► genetically defined modulation of cellular processes 5

Channelrhodopsin-2 From Ed Boyden Lab. From: Wong J, J Mech Phys Solids 2012 Jun 1; 60(6) 1158-1178 • Seven transmembrane opsin (eyespot of unicellular alga Chlamydomonas reinhardtii ) • Activated by blue light (470 nm) : the light causes a conformational change in the light sensitive molecule (retinal), which in turn causes a conformational change and the opening of the channelrhodopsin protein • Non-selective cation channel (Na + , K + , Ca 2+ , H + ): the flow of ions changes the electrical potential across the cell membrane which might, if sufficiently large, cause the neuron to fire • Widely used to depolarize neurons and generate action potential firing: very good expression in different cell hosts 6

Optogenetic control of Cav1.3 • L-type calcium channel • High Voltage Activated (HVA) • α1 (pore) + α2δ, β, γ (accessory) subunits • Therapeutic target: Cardiovascular, hormone secretion, CNS ( Parkinson’s , Alzheimer’s disease) • Drug need: Cav1.2 selectivity; state-dependent State-dependent blocker OPEN CLOSED INACTIVE Low [K + ] o High [K + ] o KIR2.3 BLUE LIGHT ChR2 DARK 7

Cav1.3 optogenetic assay Human ChR2 Human Cav1.3 ( α1,β3,α2δ1) KIR2.3 (D156A) 470nm K + K + K + K + Na + Ca 2+ K + K + K + α 2 α 1 δ 1 β 3 HEK-293 cells Na + Ca 2+ K + K + Ca 2+ Na + K + K + Ca 2+ Ca 2+ Na + Ca 2+ K + K + Ca 2+ Ca 2+ Fluo-8,NW 8

Channelrhodopsin-2 and cell based assays KEY QUESTIONS POTENTIAL ISSUES • • Is it possible to adapt the assay to the FDSS The light produced by the instrument LED µCELL optics for use in HTS? system might not have the adequate intensity for ChR2 activation • • Can ChR2 be used to depolarize cells, such The ion flux through the ChR2 might be not as HEK293, avoiding the artificial sufficient to induce membrane depolarization depolarization protocols such as KCl injection? • Does the exposure of the cells to blue light of • The membrane depolarization induced might be adequate intensity induce a ChR2 dependent not sufficient to drive the activation of cellular depolarization with subsequent transfected voltage gated channels. activation of the transfected target? • Does the ion flux through ChR2 alter the • ChR2 is not permeable to Ca 2+ in the presence detection of the transfected target? of extracellular Na + ; therefore Cav channels are ideal targets to be modulated with optogenetics, since their activity can be monitored by the use of a Ca 2+ sensitive dye GOAL Generate stable cell lines co-expressing a Voltage Gated ion channel of interest and ChR2 without altering the ion channel pharmacology 9

ChR2 induced membrane depolarization • FDSS µCELL LED efficiently activates ChR2 D156A (minimum light intensity required for wild-type ChR2 activation: 1mW/mm 2 ; Aravanis, 2007 ) ( t -off 6.9 min) Blue light (  = 480 nm; 0.013 mW/mm 2 ) 50% • Membrane depolarization repolarization half-recovered after ≈ 10 min 10

Cav1.3 half-inactivation protocol LIGHT Blue light • ChR2 Blue light • Fluo8 50% inactivated 5 - 45 min Fluo 8 dye (4 mM K + ; 2µM retinal) • Cav1.3 efficiently activated by ChR2 • 50% recovery from inactivation after 10 min INACTIVE RESTING 2 0 K + H e k 2 9 3 (v o lta g e c la m p ) 0 -2 0 V r (m V ) -4 0 T h e o r y -6 0 50% K ir 2 .3 (v o lta g e c la m p ) Fluo 8 dye K ir 2 .3 (c u rre n t c la m p ) inactivated -8 0 C a V 1 .3 (c u rre n t c la m p ) 75mM K + 0mM - 75 mM K + -1 0 0 1 1 0 1 0 0 1 0 0 0 + ] m M [ K • Cav1.3 efficiently activated by K + • 50% Cav1.3 inactivation in 16mM K + 11

State-dependent blockers with «Light protocol» ISRADIPINE dose-response @ µCELL RESTING STATE Blue light Blocker DR 5 min Fluo 8 dye (4 mM K + ; 2µM retinal) Blue light Blue light Blocker DR HALF-INACTIVATED STATE 10 min Fluo 8 dye (4 mM K + ; 2µM retinal) • Very nice Cav1.3 activation by ChR2 • State dependency well detected by Light inactivation protocol Read interval 0.1s Exp.: 0.03s; Sens.: 3 12

Light protocol vs. K + protocol vs. qPatch • “Light protocol” well suitable for state -dependent C a v 1 .3 s ta te -d e p e n d e n t blockers studies b lo c k e r p h a r m a c o lo g y L ig h t v s . K + p ro to c o l • Good correlation with classical “K + protocol” 8 (less physiological)  F /F 0 (F lu o ,8 -N W ) 6 • Good correlation with patch-clamp 4 C a v 1 .3 /C h R 2 @ q P a tc h 1 6 x 2 1 0 0 0 % R e m a in in g I C a v 1 .3 7 5 1 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4 Is ra d ip in e [M ] 5 0 2 5 R e s tin g (d a rk ): IC 50 1 3 7 n M + ): IC 50 9 5 n M R e s tin g (4 m M K 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 H a lf-in a c t (B lu e lig h t  10 m in ): IC 50 1 2 n M Is ra d ip in e [M ] + ): IC 50 1 4 n M H a lf-in a ct (1 6 m M K C lo se d (H P = -90 m V ): IC 50 3 6 2 n M H a lf-in ac t (H P = -6 0 m V ): IC 50 3 2 n M 13

Summary and conclusions “K + protocol” “Light protocol” Isradipine IC 50 qPatch 16x Literature Resting 95 nM 137 nM 362 nM 300 nM (-90mV) Half-inactivated 14 nM 12 nM 32 nM 30 nM (-50mV) RATIO 6.8 11.4 11.3 10 MAIN ACHIEVEMENTS: • FDSS µCELL optics is well suitable for ChR2 activation • A “ Light protocol ” was set up at the FDSS µCELL to study the Cav1.3 channel either in resting or inactivated state • The pharmacology of known state dependent blockers has been successfully validated, showing a good agreement with the classical “K + protocol”, patch clamp experiments and literature data HIGHLIGHTS: • First time ChR2 used for optical control of recombinant voltage-gated calcium channel assay • Physiological, robust, precise activation of Cav1.3 channel FUTURE PERSPECTIVES: • Light modulation of other voltage-gated ion channel target is ongoing 14

Aknowledgments AXXAM: Alberto di Silvio cell line generation Sara Tremolada cell line validation Jean-Francois Rolland patch-clamp Katharina Montag clonings Loredana Redaelli cell biology head Lia Scarabottolo discovery services director Stefan Lohmer overall strategies Hamamatsu team: Jean Marc d'Angelo Annamaria Mauro Laura Confalonieri Via Meucci 3 20091, Bresso (Milan, Italy) phone + 39 02 210561 fax + 39 02 2105602 www.axxam.com