Transported by light: optogenetic control of NCX1Riccardo Rizzetto , Silvia Cainarca , Viviana Agus , Lucia Rutigliano , Loredana Redaelli , Lia Scarabottolo , Andre Bazzone , Maria Barthmes , Jean-Francois Rolland
11111122

1

1. Axxam SpA, Bresso, Italy, 2. Nanion Technologies GmbH, Munich, Germany

Electrophysiological validation

Cell line selection: CPA – Fluo8NW dye
B

1.4
Response over baseline

1.2

1.1

1Na+ SWITCH PROTOCOL

Opto NCX

1.3

1.2

1.1

200 msNa+-Switch
K5-3-M11N == 10/101.8-3-

N== 5/5

1.6
1.4
1.2

1.8
1.6

FLIPRTETRA assay optimization

1.4

15,000; 20,000; 25,000 or 30,000 cells/well were seeded inblack-clear poly-D-Lys 384 MTP for 24 – 48 – 72 h. FLIPRTETRA
functional test was performed using the “Opto-NCX” protocol.
NCX1 signal was measured after the injection of 31.6 µM
KB-R7943 or 0.5 %DMSO (final concentration) in Tyrode’sbuffer (exc. 470-495nm/em.515-575). RZ’-prime calculation
was performed using as neutral control: 0.5% DMSO and31.6 µM KB-R7943 as inhibitor control, following the reported
formula. The condition 25,000 c/w for 48 h was finally selected.
Concentration-response curves of KB-R7943 and Ni2+ obtainedwith the “Opto-NCX” protocol using the HEK-293/ChR2T159C/
NCX1 and the HEK-293/ChR2T159C (mock) cell lines or theHEK-293/ChR2T159C/NCX1 cell line alone (upper + middle
and lower panels, respectively). IC50 calculation was generated
with GraphPad Prism 6 software.

1.2
1

Opto-NCX
M131.6µM KB-R7943

OPTO-NCX PROTOCOL

0.5% DMSO

Opto-NCX
Opto-NCXHEK-293 cells expressing ChR2T159C, previously generated in Axxam, were stablytransfected with NCX1 gene or with an empty vector.
After ten days of antibiotic selection, cell pools were put in Limiting Dilution
at 0.3 cells/well in 10x96 MTP. 21 days later a clone pool analysis (CPA) wasperformed 24h after replica plate in black-clear poly-D-Lys 384 MTP.
The 12 best responding positive clones and 4 mock clones were picked andexpanded. All these clones were retested at FLIPRTETRA as counted cells with both
“Na+ switch” and “Opto-NCX” protocols. The best performing clone was selected
and analyzed by manual patch clamp. Further characterization was performedon the final clone by FLIPRTETRA and SURFE2R analysis.
FLIPRTETRA Na+ switch protocol
25000 cells/well were seeded in poly-D-lysine coated, clear-bottom, black, 384well polystyrene assay plates. 48 h later, culture medium was removed andcells were incubated with 20 µl/w 0.5X Fluo-8 NW dye in Solution 1 for 45 minat room temperature. Then, cells were incubated for 20 minutes in Solution
2, containing different concentrations of antagonists; 0.1% DMSO was usedas vehicle. The activation of the NCX1 in reverse mode was finally obtained
injecting Solution 3.
FLIPRTETRA Opto-NCX protocol
25000 cells/well were seeded in poly-D-lysine coated, clear-bottom, black,384-well polystyrene assay plates. 48 h later, culture medium was removed
and cells were incubated with 20 µl/w 0.5X Fluo-8 NW dye for 45 min at room
temperature. Different concentrations of antagonists were added and incubatedfor 20 min at 37°C. The activation of the NCX1 in reverse mode is obtaineddirectly at FLIPRTETRA exciting the cells at 470-495nm for 270 sec every 0.55 sec(exc. 470-495nm/em.515-575).

Electrophysiology
24 hours before experiments, cells were seeded onto poly-D-lysine coatedglasses and placed in six-well plates in antibiotic-free medium containing
2 M all-trans-retinal. Standard whole-cell voltage clamp experiments
were performed at room temperature.
To elicit ChR2 currents, cells were continuously kept at -80 mV and blue
light stimulation was delivered by a fluorescent lamp. To isolate NCX1currents, cells were kept at -80 mV and a slow (1 s) voltage ramp from -120
to +40 mV was applied at 1 Hz; NCX1 was blocked applying
5 mM Ni2+ or 10 M KB-R7943. Data was acquired with HEKA EPC10
digitally controlled amplifier in combination with PATCHMASTER software(HEKA Electronics). The NCX1 currents were defined as the Ni2+ and KBRsensitive currents, obtained by digital subtraction.
SURFE2R -4HEK-293/ChR2T159C/NCX1 cells were cultivated, harvested, lysed and theplasma membrane was collected by partial centrifugation.
The resulting membrane vesicles were adhered to a solid supported lipid
membrane (SSM) of 3mm diameter. The ion currents through the vesicle
membrane, triggered by sequential perfusion with different buffers, weremeasured with the SURFE2R N1 device by capacitive coupling.
Solutions
Na+ Switch Protocol
Solution 1 (mM): 130 NaCl, 5 KCl, 1 MgCl2, 5 NaHCO3, 20 HEPES, pH 7.4
Solution 2 (mM): 3 CaCl2, 130 NaCl, 5 KCl, 1 MgCl2, 5 NaHCO3,20 HEPES, pH 7.4
Solution 3 (mM): 4.5 CaCl2, 130 sucrose, 5 KCl, 1 MgCl2,5 NaHCO3, 20 HEPES, pH 7.4

Opto-NCX Protocol
Solution 1 (mM): 130 NaCl, 5KCl, 2 CaCl2, 1 MgCl2, 5 NaHCO3,20 HEPES; 5 µM all-trans retinal; pH 7.4
Electrophysiology
Extracellular solution (mM): 145 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2,
10 HEPES, 10 glucose, pH 7.4
Intracellular solution (mM): 120 KCl, 5.374 CaCl2, 1.75 MgCl2,
10 HEPES, 10 EGTA, 5 Na2ATP, pH 7.2
SURFE2R
All buffers contain 30 mM HEPES (pH 7.4, NMDG) and 4 mM MgCl2. For
the experiments the proteoliposomes were equilibrated in a buffer
either containing 140 mM NaCl or
140 mM KCl. A buffer containing 140 mM KCl or 140 mM NaCl was usedduring solution exchange to establish an outward or inward directed
Na+ gradient respectively. For the activation of NCX a buffer containing
140 mM KCl and different concentrations of CaCl2 were used.

A. ChR2T159C currents induced by blue light of different
intensities.
Membrane vesicles were pre-loaded with 140 mM Na+ andflushed with 140 mM KCl to establish an outward directed Na+gradient. Then ChR2 was activated by the application of a 1 s
light pulse. Channel opening leads to the efflux of Na+ which isdetected as a fast decaying negative current. The peak current
depends on the light intensity. The inset shows the saturationof ChR2T159C peak currents with increasing light intensity.
-8-B. Polarity of ChR2T159C currents depend on the direction of theNa+ gradient.
Membrane vesicles were pre-loaded either with 140 mM
NaCl or KCl (black and red traces, respectively). Then theproteoliposomes were flushed with 140 mM KCl or NaCl (blackand red traces, respectively) to generate outward and inward
directed Na+ gradients, respectively. Finally, ChR2 was activatedby the application of a 400ms light pulse. Depending on thedirection of the Na+ gradient, Na+ influx or efflux are observed
as positive or negative currents respectively.
C. Ca2+ concentration dependence of NCX1 currents
Membrane vesicles were pre-loaded with 140 mM NaCl. Afterestablishing the outward directed Na+ gradient by flushingwith a buffer containing 140 mM KCl, NCX1 was activated by asolution containing different CaCl2 concentrations(0.2 – 500 µM). The currents observed show a negative polarity,reflecting the NCX1 1stoichiometry of 3 Na+ going out, and1 Ca2+ going in.

-1-MethodsD

1.2-3-K5C

1.4

Na+-Switch Test
Response over baseline

Response over baselineCB

IKB-R7943

INi2+

B

1.6

Na+-Switch Test Na+-Switch Test-2-A

1.811Rationale

SURFE R analysis
Opto NCX

1.3

In the present work we have generated a pure cell line stably
expressing ChR2T159c and NCX1, whose functionality has
been assessed through electrophysiology and fluorescence
techniques. We have demonstrated that activation of ChR2 byblue light is able to trigger the NCX1 reverse mode activity, thatcan be directly measured by Ca2+ sensitive fluorescent dyes.
Therefore, the “Opto-NCX” protocol represent an innovative,robust, simple and reliable strategy for studying the NCX1transporter in high-throughput screening assays.

2

1.1

1.4
Response over baseline

AConclusions

A

1.2

1

Response over baseline

FLIPRTETRA

Response over baseline

HEK-293/ChR2 cells were stably transfected with the NCX1 gene or an empty vector (mock). After ten days of antibiotic selection, cells
were put in Limiting Dilution and 21 days later a Clone Pool Analysis (CPA) was performed at FLIPRTETRA with a Ca2+-sensitive fluorescentdye (Fluo8-NW) . The presence of NCX1 was assessed using the Na+ switch and the Opto-NCX protocols (figure A, lower and upperpanel, respectively).
The figure B illustrates representative FLIPRTETRA traces recorded with both the “Opto-NCX” and the “Na+ switch” protocols (upper andlower panel, respectively).
The 12 best responding positive clones and 4 mock clones were picked, expanded and retested seeding 15,000 c/w for 24 hrs. The
Opto NCX+
classical “Na switch” and the innovative “Opto-NCX” protocols were used (left and rights panels, respectively).
1.4
The figure C shows exemplificative traces for “k5 positive” and “M1 mock” clones (K5 and M1, respectively)
incubated in Tyrode’s
modified buffer containing 0.5% DMSO or in presence of 31.6µM KB-R7943 (lower and higher traces, respectively). The clone k5 was
1.3
the final selected clone.

A. The figure A illustrates the “light-induced current” withrepresentative traces and quantitative analysis (left and rightpanels, respectively). The two components of ChR2T159C currents
are indicated as Ipeak (transient component) and Isus (sustainedcomponent).
All light-challenged cells showed a response to light (n = 15).
B. The figure B shows the average (± confidence intervals) Ni2+- andKB-R7943-sensitive currents evoked by slow voltage ramps:
INi and IKB-R (left and right panels, respectively). N is shown below thegraphs (x/x: cells showing response to blocker/total cells tested)

KB-R7943
KB-R7943
KB-R7943

2+2+NiNi
Ni2+

IC: :13IC
13µMµM
IC505050: 13 µM-6-Opto-NCX
Opto-NCX
Opto-NCX
IC: :0.55
IC
0.55mM
mM
IC505050: 0.55 mM-6-

KB-R7943
KB-R7943
KB-R7943

IC: :7.09
IC
7.09µMµM
IC505050: 7.09 µM

Chemicals
Stock solution of NiSO4 was dissolved 1 M in water; KB-R7943 wasdissolved at 100 mM in 100% DMSO.
The final concentration of DMSO was 0.5% for FLIPRTETRA, and 0.1% formanual patch clamp experiments.

KB-R7943
KB-R7943
KB-R7943

IC: :13.8IC13.8µMµM
IC505050: 13.8 µM-7--7-7-

2+2+
Ni
Ni
Ni2+

25,00025,000c/w
c/w
c/w
at25,000
at7272hrs
hrs
at 72 hrs

Opto-NCX
Opto-NCX
Opto-NCX: :0.1
IC
0.1mM
mM
IC
IC505050: 0.1 mM
2+2+
Ni
Ni
Ni2+

IC
: :20IC
20mM
mM
IC505050: 20 mM

25,00025,000c/w
c/w
c/w
at25,000
at48hrs
48hrs
at 48hrs+-Switch+NaNa
Na+-Switch-Switch25,000
25,000c/w
c/w
25,000c/w
atat48hrs
48hrs
at 48hrs

D. NCX1 transport activity generates a potential which can bedetected by ChR2T159C
Membrane vesicles were pre-loaded with 140 mM NaCl. Afterestablishing the outward directed Na+ gradient by flushingwith a buffer containing 140 mM KCl, NCX1 was activated bya solution containing 100 µM CaCl2. During NCX1 transport
activity an inside negative potential is generated. A few ms
after the raise of the NCX1 current a 1s light pulse was applied
to activate ChR2T159C. A resulting current of positive polarity
is observed, reflecting Na+ and/or H+ going in, driven by theinside negative potential which was generated by NCX1.
The inset shows the change of the ChR2T159C peak currents
with different time intervals between NCX1 and ChR2T159Cactivation. The longer the NCX1 activation, the higher the inside
negative potential, the more the ChR2T159C signal is affected.
Acknowledgements
This work was supported by the “Ministero dell’istruzione, dell’università e della ricerca” (MIUR)
and the “Bundesministerium für Bildung und Forschung” (BMBF) through the EuroTransBio
project “OPTEL” (ETB-2015-32).

Axxam SpA • OpenZone
via Meucci 3 • 20091 Bresso, Milan (Italy) • phone +39 02 210561 • fax +39 02 2105602 • www.axxam.com

The cardiac Na+-Ca2+ exchanger (NCX1) is one of the key modulatorsof the cardiomyocytes’ Ca2+ homeostasis and its reverse-mode has
been related to several disorders (e.g. heart failure). It therefore
represents an important target for both cardiac safety and drug
discovery. Robust high-throughput screening (HTS) assays arerequired to allow early stage of drug discovery, but the so far available
assays are poorly specific and involve complex solution exchange
protocols.
Our aim was to develop a new HTS-compatible strategy to trigger theNCX1 reverse-mode activity by the use of optogenetics inHEK-293 cells and to validate this approach in human induced
pluripotent stem (iPS) cells-derived cardiomyocytes assays, in order tohave a more physiological human background.
As a proof of concept, we generated a pure HEK-293 clone stably
expressing both Channelrhodopsin (ChR2) and NCX1, and we
confirmed their functionality with manual patch clamp in voltageclamp mode and standard fluorescence protocols at FLIPRTETRA,
measuring intracellular Ca2+ changes or membrane depolarization.
Our rationale is that following the ChR2 light stimulation we promoteconditions that favour the reverse-mode function of NCX1, which
can be evaluated by monitoring intracellular Ca2+. The same “OptoNCX1” protocol was used in iPS-derived cardiomyocytes co-cultured
with ChR2-expressing HEK-293 cells. The contribution of NCX1 tothe global Ca2+ cycling of the resulting syncytia was evaluated bypharmacological tools at FLIPRTETRA.
Our results indicate that ChR2 activation by blue light is successfulin triggering NCX1 reverse-mode function. This is confirmed by theeffect of specific blockers, such as KB-R7943.
Thus, we have developed a new optical tool suitable to run HTS,
looking for novel molecules acting on NCX1, and to evaluate potential
cardiotoxicity of compound, in the early phase of drug discovery
campaigns. This approach can be extended to both patient-specificand heterologous backgrounds.

500 pA

Abstract