Ion Channels:

Application Note

hERG,
NaV1.5Cells:

HEK-293,CHO
Tools:

There is no F in APC: Reliable fluoride-freerecordings on the SyncroPatch 384

SyncroPatch 384

Nanion Technologies GmbH, Munich.
SB Drug Discovery, Charles River Laboratories.

Summary
Automated patch clamp (APC) instruments are used for awide variety of applications ranging from basic research
into channelopathies and biophysical characteristics of ionchannels, through to routine cardiac safety testing. Their use
in cardiac safety screening has increased over the years

not use divalent ‘seal enhancer’ solutions, we have developed
a method that allows fluoride-free, physiological solutions to beused with good success rates. We demonstrate this using thecardiac ion channels hERG expressed in HEK293 cells (SB DrugDiscovery) and NaV1.5 expressed in CHO cells (Charles River).

and APC is now an established and accepted technique in
most, if not all, safety testing laboratories. It is well known thatfluoride is often used in the internal solution in APC experiments
to improve the seal resistance. The presence of external
calcium (or other divalent cation) further improves the sealby a mechanism thought to be due to the formation of CaF2
crystals at the interface between the pipette or micro-pore
and the cell as described in a recent patent application 1.
Even in manual patch clamp experiments, fluoride has beenused to record voltage-gated Na+ channels for over 20
years 2,3,4, despite known effects on voltage dependence ofconductance, steady-state fast inactivation 4-6 and its inhibitionof protein phosphatase 7. In addition to effects on biophysicalResultshERG expressed in HEK293 cells were recorded on theSyncroPatch 384 using fluoride-free (FF) internal solution(Figure 1A) or standard (fluoride-containing) internalsolution (Figure 1B). For hERG, a classical double voltagestep protocol was used to record hERG tail currents andwe determined the number of wells available for recording
under both conditions. Little difference in current amplitude(Figure 1C) and RSeal (Figure 1D) was observed between thetwo groups.

properties of ion channels, fluoride binds to calcium making
its use in experiments involving activation of ion channels byinternal free calcium somewhat limited, and itself activates
certain ion channels such as CFTR 8. It is also well-known thatfluoride activates G-proteins when complexed with Al3+ presentAin trace amounts 9. Fluoride is used because it improves theseal and allows stable measurements to be performed overlong periods of time 10,11. However, because there are someB

Control at start
Control at end
Full block(E4031)

Control at start
Control at end
Full block(terfenadine)

experiments where it is advantageous to use physiological,
fluoride-free internal solutions and external solution that does

0.2 nA
200 ms1(182)solution and after block with E4031 or terfenadine D
Bar graph of RSeal values for one experiment for wells
for FF and standard internal (mean ± SEM).

stternal

0.0

coFF

1.0

in ante da
rn rd
al

an example NPC-384 chip in FF and standard internal

2.0
1.5

0.5

0

recorded from an example cell using standard
internal solution. C Box plot of current amplitudes for(152)

2.5

in

recorded using FF internal solution. B hERG currents

(152)

ntrol
sta
co n
nt daro rdFFl
Full blocksta
Fu nll b dalo rdck

Figure 1: A hERG currents from an example cell(182)

DRSeal (GΩ)

Imax (nA)2FFC

Current amplitude was also stable over time and hERG-

We were also interested in investigating the effects of internal

mediated currents were almost identical at the end of thefluoride on the Vhalf of activation and inactivation of NaV1.5

recording compared with the start (Figure 1). A single additionof compound per well was performed and at the end of theexperiment, a full block concentration was used. The hERG
current was completely inhibited by a full block concentrationof either E4031 or terfenadine (Figure 1).

channels because it has been reported previously thatfluoride affects these parameters in other NaV channels 4-6,12.

There is a relatively large variability in Vhalf values reported for
NaV1.5 in the literature, ranging from -25 mV 13 to -50 mV 14.

This is presumably due to different experimental parameters
including voltage protocol, recording solutions, co-expression

Success rates (% available wells) were calculated for multiple

with β-subunits, recording temperature, holding potential, orNPC-384 chips using either standard internal solution or FF

cell expression system. For this reason, we kept all experimental

internal solution and no use of ‘seal enhancer’. Althoughparameters the same, and only changed the internal solution

success rates were better when fluoride was used, asin order to examine only the effect of fluoride on the Vhalf

expected, success rates of 34 ± 8% (n = 32 chips) wereachieved without fluoride for wells with RSeal ≥ 1GΩ at the endof the experiment, and this increased to 57 ± 9% if the qualitycontrol parameter RSeal ≥ 0.25 GΩ at the end of the experiment
was used, giving a success rate of almost 60% for completedexperiments. There was little loss of wells over time, the successof activation and inactivation of NaV1.5. Our results showthat fluoride in the internal solution causes a negative shift
in the Vhalf of activation and inactivation of NaV1.5. Figure 4
shows the activation and inactivation curves of NaV1.5. forboth conditions (standard or FF internal solution). The Vhalf ofactivation was -25.2 ± 0.5 mV (n = 241) for FF and -37.5 ± 0.4 mV

rate at the end of the experiment was similar to that at the(n = 484) for standard internal solution. A similar difference

start (Figure 2A,B).

was observed for the Vhalf of inactivation with -53.6 ± 0.6 mV(n = 165) in FF and -68.4 ± 0.4 mV (n = 728) using standard

We also used CHO cells expressing the cardiac NaV1.5

internal. This agrees with previous reports that fluoride shifts

42 ± 9% in FF versus 78 ± 5% in standard solution (Figure 2C)

values of NaV1.3 12, NaV1.7 5, and NaV1.9 4,6. The values forchannel. Using these cells, success rates for RSeal ≥ 1 GΩ werethe Vhalf of activation and inactivation to more negative

were achieved. This increased to a success rate of 55 ± 12%

Vhalf of activation and inactivation for NaV1.5 were in goodfor FF if the quality control parameter RSeal ≥ 0.25 GΩ wasagreement with the literature 13-16.

used and 86 ± 6% for standard solution (Figure 2D), a cutoff
which indicates an acceptable RSeal for screening. There waslittle loss of wells over time, the success rate at the end of theexperiment was similar as at the start.

To test whether fluoride influences pharmacology of NaV1.5

we used the known NaV channel blocker, tetracaine. This

was applied as a single concentration of compound to eachwell, followed by a full block concentration (333 μM). Using

NaV1.5-mediated currents were stable over time in botha holding potential of -120 mV, the concentration response

FF internal solution or standard internal solution (Figure 3)

curves for tetracaine in standard internal or FF internal solution

and completely inhibited by a full block concentration ofalmost exactly overlaid (Figure 5), and the IC50 values fortetracaine (333 μM).

tetracaine were not statistically significantly different (Student’st-test, P > 0.05) for 4 NPC-384 chips in standard internal and 3
NPC-384FF chips in FF internal.
As expected,the potency of inhibition increased with a more

Available wells (%)

standard internal(n = 10)fluoride-free internal(n = 32)

80
60
40
200

Available wells (%)

CB

10080

start of exp.
RSeal ≥ 1 GΩ

CHO-NaV1.5

200start of exp. end of exp.
RSeal ≥ 1 GΩ RSeal ≥ 1 GΩ

depolarized holding potential (-100 mV or -80 mV) for both80

conditions, consistent with local anesthetics preferably binding60

to the inactivated states 17-20 (data not shown, for full data set

40

please see Ref. 21).

20

RSeal ≥ 0.25 GΩ

Dstandard internal(n = 15)
fluoride-free internal(n = 17)40

100

0start of exp.

end of exp.
RSeal ≥ 1 GΩ

60

Available wells (%)

HEK-hERG

100

Available wells (%)

A

end of exp.
RSeal ≥ 0.25 GΩ

10080

Figure 2: Success rates for HEK and CHO cells in60

the presence and absence of fluoride. Shown40

are bar graphs for number of successful wells for20

HEK-hERG cells RSeal ≥ 1 GΩ (A) or RSeal ≥ 0.25 GΩ (B)0start of exp.
end of exp.
RSeal ≥ 0.25 GΩ RSeal ≥ 0.25 GΩ

and for CHO-NaV1.5 cells where RSeal ≥ 1 GΩ (C) orRSeal ≥ 0.25 GΩ (D).

Figure 3: A NaV1.5-mediated currents recorded

Aon the SyncroPatch 384 using FF internal solution

BControl at start
Control at end
Full block(tetracaine)or (B) standard internal solution. C Box plot of
current amplitudes for an example NPC-384
plate in FF and standard internal solution. The

0.5 nAcurrent amplitude after block with tetracaine is10 ms

0D

2.5(121) (132)
Imax (nA)

-1

2.0
1.5
1.0
0.5

-2

0.0

FF

-3

in
ter

Conclusions(121) (244)nalstin ante darn rdal± SEM.

RSeal (GΩ)

C

standard internal, respectively. Shown are meanFFexperiment for 121 and 244 wells for FF andcontrostl
co andnt aro rdl
FFFull blostFu an ckll b dalo rd
ckalso shown. D Bar graph of RSeal values for one

The SyncroPatch 384 is a high throughput and highly reliable
automated patch clamp device which can be used withstandard (fluoride-containing) internal solution for high successrates and long-lasting recordings. Alternatively, fluoride-freephysiological internal solution (and no external seal enhancer)

We tested different cell lines and found that similar successcan be used with good success rates, making it possible torates were achieved for CHO and HEK293 cells using FF internalperform high throughput APC recordings in physiologicalsolution. Little difference was observed for hERG currents,

solutions used in manual patch clamp. We have overcome

however, in experiments involving NaV channels, we confirmed

the challenge of low success rates and low seal resistances onhigh throughput automated patch clamp devices when using
fluoride-free physiological internal solution by implementing

a hyperpolarizing effect of fluoride on Vhalf of activation andinactivation. Although this did not affect IC50 of tetracaine, users
should be aware that fluoride causes shifts in activation andthe use of specialized consumables (NPC-384FF). This has

inactivation parameters and caution should be used whenenabled us to report success rates for fluoride-free recordings

comparing data generated in different solutions.

of approximately 60–80% for RSeal ≥ 0.25 GΩ and 40–50% for
RSeal ≥1 GΩ.

Our results further highlight the flexibility of the SyncroPatch
384 which can be used as a high throughput screening
device in standard, fluoride-containing internal solution, butalso for smaller screening projects taking advantage of the32-well mode of operation, or the use of physiological fluoridefree internal solutions which may be important for certainAB
Inactivation standard
Inactivation FFexperiments.

Standard internal
FF internal

0***0.8Vhalf (mV)

-20

0.6
0.4

***

-40

Figure 4: A Activation and inactivation IV curves for

-60

0.2

NaV1.5 in standard internal and FF internal solution

-80

tivat

at
ion

ionare shown overlaid. B The Vhalf values of activation of-20

Inaciv-60-40
Voltage (mV)ct-80

AI/Imax or G/Gmax

1.0-100

experiments or for comparison of data to manual patch clamp

Activation standard
Activation FF

484 cells in standard internal were significantly morenegative compared with 241 cells in FF internal.
The same shift to more negative Vhalf of inactivation
was also found for 728 in standard internal fluoride
compared with 165 cells in fluoride-free internal(Student’s t test, ***P < 0.001).

Standard internal
IC50 = 35.60 ± 9.41µM(n = 4 chips)
1.0

Figure 5: Pharmacology of NaV1.5 recorded inStandard internal

Tetracaine (HP -120 mV)standard and FF internal solution. Concentration
response curves for tetracaine in standard

1 nA
10 ms

Fluoride-free internal
IC50 = 41.76 ± 9.29 µM(n = 3 chips)

internal solution or FF internal solution at a holding
potential of -120 mV are shown overlaid. Example
traces in standard and FF internal solution in the0 µM tetracaine
16.65 µM tetracaine

0.6333 µM tetracaine

Normalized block

0.8

presence of tetracaine are also shown.

FF internal

0.4
0.2

0.2 nA
0.01

10100[Tetracaine (µM)]

10 ms

1000

References

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Methods
Cells
HEK-293 cells expressing hERG (SB Drug Discovery) or CHO cells
expressing NaV1.5 (Charles River) were used.
Electrophysiology
Whole cell patch clamp recordings were conductedaccording to Nanion’s standard procedure for theSyncroPatch 384. Cells were resuspended in external recording
solution and kept in the CellHotel of the SyncroPatch 384 at
12°C prior to pipetting into the NPC-384 or NPC-384FF chips
for the experiment. hERG was recorded using a double step
voltage protocol from a holding potential of -80 mV to +60 mV
for 500 ms followed by a step to -40 mV for 500 ms repeatedevery 15 s. Peak amplitude was measured at the start of thesecond voltage step. NaV1.5 was measured using a single step
protocol from -120 mV to 0 mV repeated every 10 s. Standard
internal solution contained 110 mM KF, fluoride-free internal
solution contained 110 mM K-gluconate. External solution
contained 60 mM NMDG. Please ask for more details.

vascn.2021.107125

Nanion Technologies GmbH I Ganghoferstraße 70a, 80339 Munich, Germany+49 89 2190 95-0 I info@nanion.de I www.nanion.de