Application Note

Channels:
VRAC
Cells:
Keratinocytes (HaCaT)
Tools:Patchliner,
SyncroPatch 384

The role of LRRC8 in the hypotonic stressresponse of human keratinocytes
The electrophysiology team at Nanion Technologies GmbH, Munich, in collaboration
with BRAIN Biotech AG and TU Darmstadt
Cultured human keratinocytes (HaCaT) were kindly provided by BRAIN Biotech AG

Summary

Results

The human skin is constantly exposed to various stress

LRRC8A expressed in wild type HaCaT keratinocytes wasfactors such as temperature changes, mechanical stress,

activated by hypotonic solution and blocked by DIDS on

different humidity levels, air pollution or radiation. Thesethe Patchliner. In contrast, no activation by hypotonicfactors can have a tremendous impact on the skin andsolution was observed in the LRRC8A-/- knockout HaCaT

can contribute to barrier disruption and inflammation,

keratinocytes (Figure 1).

dry and fragile skin as well as premature ageing1. Recent

AWTI (nA)esses2. The function of LRRC8 has been characterized inhuman keratinocytes and in the native human epidermis

3entiation in a recent patent4.
LRRC8A (also named SWELL1) has been identified as thefirst essential component of VRACs in various cell types5,6.

-100

-50

CLRRC8A-/-

and a C-terminal domain containing up to 17 leucine-tion differs between cell types and influences VRAC properties such as inactivation kinetics, voltage-dependence
and selectivity of the transported osmolyte9-13. The generation of LRRC8A-/- knockout HaCaT keratinocytes has
provided evidence for the essential function of LRRC8A inhypotonic stress response of human keratinocytes3.
In this Application Note we show electrophysiological
data from WT and LRRC8A-/- knockout HaCaT keratinocytes which corroborate the essential function of LRRC8Ain keratinocytes.
Download more Application Notes from www.nanion.de

2150100
Voltage (mV)005

Isotonic

4

Hypotonic

3100 µM DIDS

2100

200

300

400500Time (s)D4Hypotonic 100 µM
Isotonic
320 mOsm 234 mOsm DIDS

321rich repeats7,8. Together with four additional LRRC8 familycomplexes5,6. Interestingly, the LRRC8 subunit composi-0-2LRRC8A is composed of four transmembrane domainsmembers (LRRC8B-E) it assembles into hetero-hexameric

2-1

Hypotonic 100 µM
234 mOsm DIDS

331I (nA)novel molecular target to modulate keratinocyte differ-Isotonic
320 mOsm

44(VRACs) in a plethora of different physiological proc-

and the LRRC8 ion channel has been proposed to be aB

I+100 mV (nA)tant role of LRRC8 volume regulated anion channels

Isotonic
Hypotonic
100 µM DIDS

5I+100 mV (nA)advances in different research areas point to an impor-

1-100-50-1-2050100Voltage (mV)

0050100
150Time (s)200250Figure 1: Typical recordings from WT and LRRC8A-/- KO HaCaT keratinocytes.
A LRRC8 current was recorded using a voltage ramp protocol from -100
mV to +100 mV over 200 ms. Hypotonic solution had an osmolarity of 234mOsm, isotonic solution had an osmolarity of 320 mOsm. B Timecourse of
the experiment shown in A, after addition of hypotonic solution, there wasa large increase in current at both +100 mV and -100 mV (not shown). TheLRRC8 current was blocked by DIDS. C There was no activation of LRRC8
current in the knockout keratinocytes recorded on the Patchliner using avoltage ramp protocol. D Corresponding timecourse of the experiment,
there was no activation of LRRC8 current and no block of the current byDIDS.

Application Note
In 11/11 WT keratinocytes, an increase in current at

A: WT-100 mV from -0.2 ± 0.02 nA to -1.2 ± 0.1 nA, an increaseof over 400%, and at 100 mV from 0.7 ± 0.1 nA to 2.8 ± 0.3
nA, an increase of 360%, was observed (Figure 2, left). Thecurrent at -100 mV and +100 mV was blocked by DIDS in

1 nA

11/11 cells tested. In LRRC8A-/- keratinocytes, 7/8 cells did

200 msWT (n = 13)

6upon addition of DIDS (Figure 2, right), although this was4

not statistically significant.

2-0.8-1.2-120 -80 -40 0-2

40 80 120
Vm (mV)on the high throughput automated patch clamp device,
Isotonic Hypotonic

100 µM
DIDS-0.4

the SyncroPatch 384. Figure 4 shows the hypotonic activated currents measured using a voltage ramp protocol
and partial reversal following washout in isotonic solution.
AB 0.5
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4

-0.8
-1.2

**

**2WT and LRRC8A-/- HaCaT keratinocytes were also used

1040 80 120
Vm (mV)*
Figure 2: Effect of hypotonic solution on WT and KO keratinocytes on thePatchliner. Average current size in isotonic, hypotonic and in DIDS for WT (left)and LRRC8A-/- (right) keratinocytes. **P < 0.05; **P<0.001, paired Students ttest.

LRRC8 was also measured using a voltage step protocol on the Patchliner (Figure 3). LRRC8 expressed in HEKIsotonic
Hypotonic Washout(320 mOsm) (277 mOsm) (320 mOsm)

-0.2
-0.3
-0.4
-0.5
-0.6
-0.7
-0.8
-0.9
-1.00100 200 300 400 500 600 700 800
Time (s)Cat positive potentials above 100 mV (Figure 3A). In contrast, LRRC8A cells did not show any activation at any of-/-the voltages tested in 5/5 cells (Figure 3B, right).

Download more Application Notes from www.nanion.deWT
LRRC8A-/-

0.3
0.2
0.1

(65)

0.0
-0.1
-0.2

-0.4DIsotonic
Hypotonic
Washout (isotonic)

inactivation at positive potentials6. On the Patchliner,using hypotonic solution and also displayed inactivation

(49)

-0.3
WT
LRRC8A-/-and HCT116 cells has been shown to display prominent
LRRC8 expressed in HaCaT keratinocytes was activated

0.4
Hypotonic-activated Current (nA)

I+100 mV (nA)

-0.4

20100 µM
DIDS*

4ControlHypotonic

Current (nA)

I-100 mV (nA)

Isotonic Hypotonic

I-100 mV (nA)

I+100 mV (nA)1***

Figure 3: Effect of hypotonic solution on WT and LRRC8A-/- HaCaT
keratinocytes using a voltage step protocol on the Patchliner. A Traces froma representative cell in isotonic (left) and hypotonic (right) conditions. B
Current voltage-plot for an average of 13 WT cells (left) and 5 LRRC8A-/- cells(right).

320

*

**

**
30-120 -80 -40 * 0*-2* *LRRC8A-/-***

Current (nA)WT

LRRC8A-/- (n = 5) 6*

IVRAC (nA)rent) and a moderate decrease in current was observed

BIVRAC (nA)

not respond to hypotonic solution (<100% increase in cur-0 nA

0 nA
0.5 nA

100 msFigure 4: Hypotonic-activated current in WT and LRRC8A-/- HaCaT
keratinocytes on the SyncroPatch 384. A Current amplitude at 100 mV (top)and -100 mV (bottom) from WT (dark blue) and LRRC8A-/- (light blue) HaCaT
keratinocytes in isotonic (grey region), hypotonic (light blue region) andfollowing washout (isotonic; grey region) is shown. B Average hypotonicactivated current amplitude at +100 mV and -100 mV for WT (dark blue) andLRRC8A-/- (light blue) cells is shown as a bar graph, number of cells shown inbrackets. The hypotonic-activated current was significantly larger in WT vs.
LRRC8A-/- cells (P<0.05, unpaired Student’s t test).

Application NoteA

col on the SyncroPatch 384 (Figure 5). As shown on themV (Figure 5B). In contrast, LRRC8A-/- cells showed little
activation upon addition of hypotonic solution (Figure5C). The hypotonic-activated current was significantly
larger in WT vs. LRRC8A-/- cells for an average of 112 andIsotonic
Hypotonic0.8200C

400

Current (nA)

600
Time (s)

800

1000

1.4Current (nA)

1.2
1.0
0.8
0.6

200

400

600
Time (s)

800

1000

D
Hypotonic-activatedCurrent (nA)

C0.5 nA
400200 ms
0.5(112)WT
LRRC8A-/-

0.4
0.3
0.2
0.1
0.0

1000 1200

200 msD

Isotonic
Hypotonic
100 µM NPPB

0.8

200

0.2 nA

200 msFigure 6: Hypotonic-activated current in WT and LRRC8A-/- HaCaT
keratinocytes on the SyncroPatch 384 and block by NPPB. A Average
current amplitude of 108 cells elicited using a voltage step to +100 mVin isotonic (grey) and hypotonic (blue; 256 mOsm) solution of WT HaCaT
keratinocytes was blocked by NPPB (dark grey). B Example traces from aWT cell in isotonic (black) and hypotonic (blue) solution and block by NPPB
(red). C Average current amplitude of 103 cells elicited using a voltage
step to +100 mV in isotonic (grey) and hypotonic (blue; 256 mOsm) solutionof LRRC8A-/- HaCaT keratinocytes. D Example traces from a LRRC8A-/- cell inisotonic (black), hypotonic (blue) solution and NPPB (red).

0.8200400 600 800
Time (s)0.2 nA

1.0

1.20.61000 1200

1.20.6

1.0

600 800
Time (s)A(91)

Figure 5: Hypotonic-activated current in WT and LRRC8A-/- HaCaTkeratinocytes on the SyncroPatch 384 using a voltage step protocol. A
Current amplitude elicited using a voltage step to +100 mV in isotonic(grey) and hypotonic (blue) solution of WT HaCaT keratinocytes. B Example
traces from a WT cell in isotonic (black) and hypotonic (blue) solution. C
Current amplitude elicited using a voltage step to 100 mV in isotonic (grey)
and hypotonic (blue) solution of LRRC8A-/- HaCaT keratinocytes. D Average
hypotonic-activated current amplitude at +100 mV for WT (dark blue) andLRRC8A-/- (light blue) keratinocytes is shown as a bar graph, number of cells
shown in brackets. The hypotonic-activated current was significantly largerin WT vs. LRRC8A-/- cells (P<0.05, unpaired Student’s t test).

WT(n = 119)32

Control
Hypotonic

1*-120 -80 -40
*

*

*

*

*-1

B*
*

*

*

*

*LRRC8A-/(n = 140)

32Control
Hypotonic
1*40

80 120 -120 -80 -40***Vm (mV)*-1*

Current (nA)

1.4B

1.0

Current (nA)AIs
(3 oto20 nm icOsmH)(2 ypo77 tom ni
O csHy m)(2 po56 tm oni
O csm)
W(3 as20 hom utOsm)

91 cells, respectively (P<0.05, Student’s t test; Figure 5D).

1.2

0.6

Current (nA)displayed inactivation at positive potentials above 100

Current (nA)Patchliner above, LRRC8 expressed in HaCaT keratinocytes was activated using hypotonic solution and alsoBIs
(3 oto20 nm icOsmH)(2 ypo77 tom niO csHy m)(2 po56 tm oniO csm)
10N 0PP µM
BLRRC8 was also measured using a voltage step proto-

*

*40*

**

*80 120Vm (mV)

Figure 7: Effect of hypotonic solution on WT and LRRC8A-/- HaCaT
keratinocytes at different voltages. A Current voltage-plot for an averageof 119 WT cells. B Current voltage-plot for an average of 140 LRRC8A-/- cells.

The Patchliner and SyncroPatch 384 are automatedTheHaCaT

patch clamp devices for recording from up to 8 or 384

keratinocytes was partially blocked by the chloride

hypotonic-activatedcurrent

fromWTcells, respectively. Both devices were used to record-/-channel blocker, NPPB (Figure 6) but not LRRC8A

LRRC8 from keratinocytes in response to hypotonic

keratinocytes (Figure 6). In both WT and LRRC8A-/- kerati-stress. LRRC8A-E have been identified as volume-

nocytes a moderate, but statistically significant, current

regulated anion channels in a number of cell types5,6,

was activated at different voltages on the SyncroPatch

which assemble into hetero-hexameric complexes. Cryo-384 upon addition of hypotonic solution (Figure 7). ThisEM structures have also recently confirmed the formation

current was more prominent in WT keratinocytes.

of LRRC8 ion channel pores14-16. The LRRC8A-/- knockout
HaCaT keratinocytes were created to investigate thefunction of LRRC8 in the hypotonic stress response ofhuman keratinocytes.

Download more Application Notes from www.nanion.deApplication NoteIn

previousstudies,

LRRC8activitywasmeas-a lower osmolarity of the hypotonic solution compared

ured, as well as cell volume changes indirectlywith experiments on the SyncroPatch 384. This is because

by using the fluorescent halide-sensitive YFP and thethe SyncroPatch uses a well-based chip so that addition

volume-sensitive calcein-AM dye3. In this study weof a new solution mixes 1:1 with the solution already pre-recorded LRRC8A from WT HaCaT keratinocytes which

sent in the well. Therefore, we observed a larger effect

was activated by hypotonic solution and blocked by theon current amplitude with a faster time of onset on thebroad-spectrum chloride channel blockers, DIDS or NPPB,

Patchliner compared with the SyncroPatch 384.

on both the Patchliner and SyncroPatch 384. No LRRC8
current was recorded by addition of hypotonic solution

In conclusion, the Patchliner and SyncroPatch 384 auto-of the LRRC8A HaCaT keratinocytes in 7/8 cells tested onmated patch clamp instruments can be used to record

the Patchliner, and a much smaller hypotonic-activated

VRAC channels in keratinocytes which, in combination

current was detected in the LRRC8A cells comparedwith WT and KO keratinocytes, can be used to eluci-

with WT recorded on the SyncroPatch 384. On the Patch-date the physiological and pathological role of VRAC

liner, the microfluidic design of the chip ensures thatchannels in various disorders including inflammatory skincomplete exchange of the solution occurs resulting indiseases, ischemia and even cancer.

References

Cell culture-/--/-

1. Del Rosso, J., et al., 2016. J. Clin. Aesthet. Dermatol. 9(4, Suppl. 1): S2-S8
2. Chen, L., et al., 2019. Biol. Chem. 400(11): 1481-1496.
3. Trothe, J., et al., 2018. Exp. Dermatol. 27(12): 1352-1360
4. Ertongur-Fauth, T., et al., 2019. 1. WO2019158696 - The volume-regulated anion channel protein LRRC8A for use in altering keratinocyte

harvestedaccording to Nanion’s standard cell culture protocols.
Electrophysiology
Whole cell patch clamp recordings were conducted

osmolarity of 234 mOsm were prepared (osmolarity was

6. Voss, F.K., et al., 2014. Science. 344(6184): 634-638

adjusted using mannitol). On the Patchliner, the

7. Abascal, F. & Zardoya, R. 2012. Bioessays. 34: 551–560

hypotonic

8. Kubota, K., et al., 2004. FEBS Lett. 564: 147-152

solution

waspipetted

onto

thecells,completely exchanging the external solution resulting in

9. Lutter, D., et al., 2017. J. Cell Sci. 130: 1122-1133

a final osmolarity of 234 mOsm. On the SyncroPatch 384,

10. Planells-Cases, R., et al., 2015. EMBO J. 34(24): 2993-3008

hypotonic solution was added either once, mixing with

11. Schober, A.L., et al., 2017. J. Physiol. 595.22 : 6939–6951

the isotonic solution present in the well resulting in a final

12. Syeda, R., et al., 2016. Cell. 164(3): 499–511

osmolarity of 277 mOsm, or twice resulting in a final

13. Ullrich, F., et al., 2016. JBC. 291(33): 17040 –17048

osmolarity of 256 mOsm. A voltage ramp from -100 mV to

14. Deneka, D., et al., 2018. Nature. 558(7709):254-259

100 mV over 200 ms from a holding potential of -50 mV

15. Kauya, G., et al., 2018. Nat. Struc. Mol. Biol. 25(9): 797-804

was used on the Patchliner or from -100 mV to 100 mV

16. Kefauver, J.M., et al., 2018. eLife. 10;7:e38461

over 500 ms from a holding potential of -40 mV on theSyncroPatch 384. Current at -100 mV and +100 mV wasCollaboration partner
We thank the BRAIN Biotech AG (Dr. Torsten Fauth, UnitUniversity

andosmolarity of 320 mOsm and hypotonic solution with an

5. Qiu, Z., et al., 2014. Cell. 157: 447–458

Technicalcultured

Patchliner and SyncroPatch 384. Isotonic solution with an

jsf?docId=WO2019158696. Accessed 15.12.21.

andwereaccording to Nanion’s standard procedures for thedifferentiation. https://patentscope.wipo.int/search/en/detail.

BioActives

Keratinocytes

Performance

Biologicals)

Darmstadt(Dr.

andOlivertheRauh,

Membrane Biophysics) for providing cells and technicalexpertise.

used for analysis. Current-voltage relationships wereconstructed using a voltage step protocol from -120 mV
for 840 ms increasing in 20 mV steps to 120 mV from aholding potential of -50 mV (Patchliner) or -40 mV(SyncroPatch 384). Current amplitude was measured at
the start of the voltage step. For pharmacology

Methods

experiments using a voltage step protocol, a step from aholding potential of -40 mV to +100 mV was repeatedCells

WT and LRRC8A-/- HaCaT keratinocytes were kindly

every 15 s. All data are expressed as mean ± SEM.

provided by BRAIN Biotech AG.
Nanion Technologies GmbHphone + 49 89 219-095-0

Ganghoferstr. 70A,

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960