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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
