Viviana Agus Axxam S.p.A., Milano, Italy

Optogenetics: A Bright Future for

Voltage Gated Ion Channels

Presentation Summary

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• 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

FDSS μCELL DEMO @ AXXAM

Ca2+ assay (fluorescent dyes,

luminescent photoprotein)

• 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

• Cav1.3 (ChR2)

Fluo8 LED (blue light)

MPdye LED (green light)

Hybrid Camera

(Fluo & Lumi)

3

Optogenetics: overview

4

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

Rhodopsins: Light-gated ion channels

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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

• 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

From: Wong J, J Mech Phys Solids 2012 Jun 1; 60(6) 1158-1178

From Ed Boyden Lab.

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Channelrhodopsin-2

CLOSED OPEN INACTIVE

State-dependent blocker

• 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

Optogenetic control of Cav1.3

KIR2.3 Low [K+]o High [K+]o

ChR2 DARK BLUE LIGHT

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Cav1.3 optogenetic assay

α1

α2

δ1 β3

Na+ K+

Ca2+

Ca2+

Na+

K+ Na+

K+

Na+

K+ K+ Ca2+

Ca2+ Ca2+

Ca2+

Ca2+

Fluo-8,NW

K+

K+

K+

K+

K+ K+

K+

K+

Human

KIR2.3

Ca2+

ChR2

(D156A)

Human Cav1.3

(α1,β3,α2δ1)

HEK-293 cells

470nm

8

KEY QUESTIONS

• Is it possible to adapt the assay to the FDSS

µCELL optics for use in HTS?

• Can ChR2 be used to depolarize cells, such

as HEK293, avoiding the artificial

depolarization protocols such as KCl

injection?

• Does the exposure of the cells to blue light of

adequate intensity induce a ChR2 dependent

cellular depolarization with subsequent

activation of the transfected target?

• Does the ion flux through ChR2 alter the

detection of the transfected target?

Channelrhodopsin-2 and cell based assays

POTENTIAL ISSUES

• The light produced by the instrument LED

system might not have the adequate intensity

for ChR2 activation

• The ion flux through the ChR2 might be not

sufficient to induce membrane depolarization

• The membrane depolarization induced might be

not sufficient to drive the activation of

transfected voltage gated channels.

• ChR2 is not permeable to Ca2+ in the presence

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

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ChR2 induced membrane depolarization

• FDSS µCELL LED efficiently

activates ChR2 D156A

(minimum light intensity required for wild-type

ChR2 activation: 1mW/mm2; Aravanis, 2007)

Blue light ( = 480 nm; 0.013 mW/mm2)

• Membrane depolarization

half-recovered after ≈ 10 min

(t-off 6.9 min)

50%

repolarization

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Cav1.3 half-inactivation protocol

50%

inactivated

• Cav1.3 efficiently activated by ChR2

• 50% recovery from inactivation after 10 min

Fluo 8 dye

(4 mM K+; 2µM retinal)

Blue light

5 - 45 min

Blue light • ChR2

• Fluo8

LIGHT

K+

Fluo 8 dye 50%

inactivated

• Cav1.3 efficiently activated by K+

• 50% Cav1.3 inactivation in 16mM K+

75mM K+ 0mM - 75 mM K+

INACTIVE RESTING

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1 1 0 1 0 0 1 0 0 0

-1 0 0

-8 0

-6 0

-4 0

-2 0

0

2 0

[ K+

] m M

Vr (

mV

)

R T /z F = 5 2 .2 2

K ir2 .3 C u rre n t C la m p

R T /z F = 5 4 .7 6

K ir2 .3 V o lta g e C la m p

T h e o ry

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

R T /z F = 5 8 .7 3

R T /z F = 5 8 .9 0K ir 2 .3 (c u rre n t c la m p )

K ir 2 .3 (v o lta g e c la m p )

C a V 1 .3 (c u rre n t c la m p )

T h e o r y

H e k 2 9 3 (v o lta g e c la m p )

State-dependent blockers with «Light protocol»

ISRADIPINE dose-response @ µCELL

Read interval 0.1s

Exp.: 0.03s; Sens.: 3

RESTING STATE

Fluo 8 dye

(4 mM K+; 2µM retinal)

Blue light

10 min

Blue light Blocker DR

HALF-INACTIVATED STATE

Fluo 8 dye

(4 mM K+; 2µM retinal)

Blue light

5 min

Blocker DR

• Very nice Cav1.3 activation by ChR2

• State dependency well detected by

Light inactivation protocol

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Light protocol vs. K+ protocol vs. qPatch

• “Light protocol” well suitable for state-dependent

blockers studies

• Good correlation with classical “K+ protocol”

(less physiological)

• Good correlation with patch-clamp

1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5

0

2 5

5 0

7 5

1 0 0

Is ra d ip in e [M ]

% R

em

ain

ing

IC

av

1.3

C lo s e d (H P = -9 0 m V )

In a c tiv a te d (H P = -6 0 m V )

I C 5 0 = 3 6 2 n M

I C 5 0 = 3 2 .2 n M

C a v 1 .3 /C h R 2 @ q P a tc h 1 6 x

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 0 -1 0 1 0 -9 1 0 -8 1 0 -7 1 0 -6 1 0 -5 1 0 -4

0

2

4

6

8

C a v 1 .3 s ta te -d e p e n d e n t

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

Is ra d ip in e [M ]

F

/F0

(F

luo

,8-N

W)

1.369e-007

9.502e-008

1.146e-008

1.746e-008

R e s tin g (D a rk )

H a lf- in a c tiv a te d (B lu e L ig h t = > 1 0 m in )

R e s tin g (4 m M K+

)

H a lf- in a c tiv a te d (1 6 m M K+

)

IC 5 0 1 3 7 n M (R e s tin g ; lig h t)

IC 5 0 9 5 n M (R e s tin g ; K+)

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

H a lf- in a c t (B lu e lig h t 10 m in ): IC 5 0 1 2 n M

H a lf- in a c t (1 6 m M K+): IC 5 0 1 4 n M

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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

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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