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Optogenetics: a Bright Future for Voltage Gated Ion Channels

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Optogenetics: a Bright Future for Voltage Gated Ion Channels 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 μCELL DEMO @ AXXAM 2+ Ca 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: • PPARα, PPARδ (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+, Ca2+, 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 KIR2.3 High [K ]o DARK ChR2 BLUE LIGHT 7 Cav1.3 optogenetic assay Human ChR2 Human Cav1.3 KIR2.3 (D156A) (α1,β3,α2δ1) K+ K+ 470nm + K K+ Na+ Ca2+ + K+ K K+ α2 α1 δ1 β3 HEK-293 cells + K+ K+ Na Ca2+ Ca2+ + Na+ K K+ Ca2+ Ca2+ Na+ Ca2+ K+ K+ Ca2+ Ca2+ 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 Ca2+ 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 Ca2+ 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/mm2; Aravanis, 2007) (t-off 6.9 min) Blue light ( = 480 nm; 0.013 mW/mm2) 50% repolarization • Membrane depolarization half-recovered after ≈ 10 min 10 Cav1.3 half-inactivation protocol • ChR2 LIGHT Blue light 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 0 H e k 2 9 3 (v o lta g e c la m p ) -2 0 ) V m -4 0 ( R T /z F = 5 2 .2 2 T h e o r y r R T /z F = 5 4 .7 6 V -6 0 R T /z F = 5 8 .7 3 K ir 2 .3 (v o lta g e c la m p ) R T /z F = 5 8 .9 0 50% K ir 2 .3 (c u rre n t c la m p ) Fluo 8 dye -8 0 + C a V 1 .3 (c u rre n t c la m p ) inactivated 0mM - 75 mM K+ 75mM K -1 0 0 1 1 0 1 0 0 1 0 0 0 + [ K ] m M K ir2 .3 V o lta g e C la m p + • Cav1.3 efficiently activated by K T h e o ry + K ir2 .3 C u rre n t C la m p • 50% Cav1.3 inactivation in 16mM K H E K 2 9 3 W T V o lta g e C la m p C a v 1 .3 C lo n e K 2 .8 C u rre n t C la m p 11 State-dependent blockers with «Light protocol» ISRADIPINE dose-response @ µCELL RESTING STATE Blocker DR Blue light 5 min Fluo 8 dye (4 mM K+; 2µM retinal) Blue light Blocker DR Blue light 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)R e s tin g (D a r k ) ) H a lf-in a c tiv a te d (B lu e L ig h t = > 1 0 m in ) W 6 N - + 8 • Good correlationR e s tin g (4 withm M K patch) -clamp , o + 4 H a lf-in a c tiv a te d (1 6 m M K ) u l F ( 0 2 C a v 1 .3 /C h R 2 @ q P a tc h 1 6 x F / F IC 1 3 7 n M (R e s tin g ; lig h t) 1 0 0 5 0 0 3 . 1 v a 7 5 + C -1 0 -9 -8 -7 -6 -5 -4 I IC 9 5 n M (R e s tin g ; K ) 1 0 1 0 1 0 1 0 1 0 1 0 1 0 5 0 g n Is ra d ip in e [M ] i n 5 0 i a m e R 2 5 R e s tin g (d a rk ): IC 5 0 1 3 7 n M + % R e s tin g (4 m M K ): IC 5 0 9 5 n M 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 1 0 m in ): IC 5 0 1 2 n M Is ra d ip in e [M ] + H a lf-in a c t (1 6 m M K ): IC 5 0 1 4 n M C lo s e d (H P = -9 0 m V ): IC 5 0 3 6 2 n M H a lf-in a c t (H P = -6 0 m V ): IC 5 0 3 2 n M 1.369e-007 9.502e-008 13 1.146e-008 1.746e-008 C lo s e d (H P = -9 0 m V ) I C 5 0 = 3 6 2 n M In a c tiv a te d (H P = -6 0 m V ) I C 5 0 = 3 2 .2 n M Summary and conclusions + Isradipine IC50 “K protocol” “Light protocol” 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 .
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