Optogenetics: A Bright Future for Voltage Gated Ion Channels

Channelrhodopsin-2 (ChR2) is a light-activated microbial cation channel

which can be used to depolarize neurons through the incidence of blue light

(470 nm). The possibility to optically control the plasma membrane voltage

opens new and interesting perspectives for the characterization of

voltage-gated ion channels and the search for modulators.

Proof of principle studies have been performed to verify the applicability

of this tool for the development of cell based assays in High Throughput

Screening (HTS) platforms as microplate readers.

An HEK-293 cell line, which stably co-expresses the human voltage-gated

calcium channel hCav1.3 and the inward rectifi er hKir2.3 channel, was

over-transfected with a ChR2 carrying a single amino acids mutation. This

latter results in a prolonged lifetime of the conducting state of ChR2 and

in a reduced light power requirement for its activation.

A protocol of light stimulation of ChR2 and record of calcium ion infl ux

through Cav1.3 with the use of a calcium-sensitive fl uorescent dye (Fluo8)

was tested in the Hamamatsu FDSSµcell. A well-known Cav1.3 blocker,

Isradipine, tested in the resting and the partial inactivated Cav1.3 states,

was used to confi rm the pharmacological profi le.

Data obtained for the ChR2/hCav1.3 cell line by light stimulation have

been also compared to the extracellular potassium stimulus and to patch-

clamp to cross-check their reliability.

Key-questions of the study

Is the blue light produced by the FDSSµcell LED intense enough

to activate ChR2? ➔ Can the light protocol be runned with the

FDSSµcell?

Is the activation of ChR2 suffi cient to induce cell membrane depola-

rization? ➔ Can the ChR2 be used to depolarize cells avoiding the

irreversible depolarization protocols using KCl injection?

Is the resulting membrane depolarization suffi cient to activate the

transfected voltage-gated channel Cav1.3?

Can the cation fl ux through ChR2 disturbe the detction of Ca2+

fl ux through the transfected target Cav1.3?

Dihydropyridine

Phenylalkylamine

Benzothiazepin

Membrane repolarizationTime course of membrane repolarization after a fi rst blue light pulse (MPdye)

Cell membrane repolarization

%

50 % repolarization

Ca++ infl ux through Cav1.3Time course of signal recovery after a fi rst blue light pulse (Fluo 8 NW)

First pulse Second pulse

∆F/F

0

50 % inactivated

Cav1.3 state-dependent blocker pharmacology Light vs. K+ protocol

Cav1.3/ChR2 @ qPatch 16x

Resting (dark): IC50 137 nMResting (4 mM K+): IC50 95 nMHalf-inact (Blue light�➔ 10 min): IC50 12 nMHalf-inact (16 mM K+): IC50 14 nM

Closed (HP = -90 mV): IC50 362 nMHalf-inact (HP = -60 mV): IC50 32 nM

Isradipine IC50 “K+ protocol” “Light protocol” qPatch 16x LiteratureResting 95 nM 137 nM 362 nM 300 nM (-90 mV)

Half-inactivated 14 nM 12 nM 32 nM 30 nM (-50 mV)

RATIO 6.8 11.4 11.3 10

10- 9 10- 8 10- 7 10- 6 10- 50

25

50

75

100

Isradipine [M]Isradipine [M]

% R

emai

ning

ICav

1.3

10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 4

0

2

4

6

8

∆ F/

F0 (F

luo,

8-N

W)

13

10- 9 10- 8 10- 7 10- 6 10- 50

25

50

75

100

Isradipine [M]Isradipine [M]

% R

emai

ning

ICav

1.3

10- 1 0 10- 9 10- 8 10- 7 10- 6 10- 5 10- 4

0

2

4

6

8

∆ F/

F0 (F

luo,

8-N

W)

13

Electrode

Micropipette

Cell membrane Na+ channel

Low [K+]O

State-dependent blocker

CLOSED

Resting

Resting

HumanKIR2.3

HEK-293 cells

Fluo-8,NW

ChR2(D156A)

Human Cav1.3(α1, β3, α2δ1)

Depolarized

Depolarized

INACTIVEOPEN

DARK

High [K+]O

BLUE LIGHT

Vm (mV)

-2.3 x 10-10

-60 -40 -20 20 40

Char

ge

ChR2

KIR2.3

Fluo 8 dye(4 mM K+; 2 µM retinal)

Blue lightBlue light Blocker DR

10 min

Fluo 8 dye(4 mM K+; 2 µM retinal)

Blue lightBlocker DR

5 min

Ca2+

Cell membrane

Vm (mV)

Vh = -39.52 mV

-100 -50

1.0

0.5

0.00

I/Im

ax

0 mV3000 ms

100 ms

-100 mV

-20 mV

Hamamatsu Photonics Italia srlStrada della Moia 1 int 6

20020 Arese, Milano (Italy)

Annamaria Mauro Laura Confalonieri

Axxam SpAVia Meucci 3

20091 Bresso, Milano (Italy)

Loredana Redaelli Jean-Francois Rolland Lia Scarabottolo Viviana Agus Stefan Lohmer

Hamamatsu Photonics FranceParc Du Mulin de Massy

19 Rue du Saule Trapu

91300 Massy (France)

Jean Marc D’Angelo

150421_ham_poster_fdss_optogen_1000x1400mm.indd 1 21.04.15 16:58