The role of LRRC8 in the hypotonic stress response of human keratinocytes
Maria Giustina Rotordam1, Andràs Horvàth1, Torsten Fauth2, Claudia Buerger3, Deborah-Désirée Scheub4, Oliver Rauh4,Alison Obergrussberger1
1Nanion Technologies GmbH, Munich, Germany; 2BRAIN Biotech AG, Zwingenberg, Germany; 3Department of Dermatology, Clinic of the Goethe-University, Frankfurt

am Main, Germany; 4Plant Membrane Biophysics, Technische Universität Darmstadt, Darmstadt, Germany

VRAC channels regulate cell volumeIntroduction
The human skin is constantly exposed to various stress factors such as temperaturechanges, mechanical stress, different humidity levels, air pollution or radiation.
These factors can have a tremendous impact on the skin and can contribute tobarrier disruption and inflammation, dry and fragile skin as well as prematureageing (Rosso et al., 2016). Recent advances in different research areas point toan important role of LRRC8 volume regulated anion channels (VRACs) in a plethoraof different physiological processes (Chen et al., 2019). The function of LRRC8 has
been characterized in human keratinocytes and in the native human epidermis(Trothe et al., 2018) and the LRRC8 ion channel has been proposed to be a novel
molecular target to modulate keratinocyte differentiation. LRRC8A (also namedSWELL1) has been identified as the first essential component of VRACs in various
cell types (Qui et al., 2014; Voss et al., 2014). LRRC8A is composed of fourtransmembrane domains and a C-terminal domain containing up to 17 leucinerich repeats. Together with four additional LRRC8 family members (LRRC8B-E) itassembles into hetero-hexameric complexes (Qiu et al., 2014; Voss et al., 2014). TheLRRC8 subunit composition differs between cell types and influences VRAC properties such as inactivation kinetics, voltage-dependence and selectivity of thetransported osmolyte. The generation of LRRC8A-/- knockout HaCaT keratinocytes
has provided evidence for the essential function of LRRC8A in hypotonic stressresponse of human keratinocytes (Trothe et al., 2018).

Hypoosmotic conditions result in cell swelling,which is counteracted by regulatory volume
decrease (RVD) to prevent necrotic cell
death. RVD involves activation of volumeregulated anion channels (VRACs), whichallow efflux of chloride (Cl-). Subsequent effluxof osmotically driven water restores the initialcell volume.

VRAC

Adapted fromEl-Chami et al., 2014

LRRC8A mediates VRAC activity and RVD
AVRAC

halide-sensitive YFPB
VRAC

Calcein-AM dye

Abascal & Zardoya, 2012

(A) Keratinocytes were engineered to express the I- sensor hsYFP. Hypotonic stimulation ofHaCaT WT, but not of HaCaT LRRC8A-/- triggered an I--dependent quenching of hsYFPfluorescence. VRAC activity was drastically reduced in LRRC8A-deficient HaCaTs as well as
NHEKs. (B) Keratinocytes were loaded with volume-sensitive dye calcein-AM. Reduction ofcalcein fluorescence upon hypotonic stimulation of HaCaT LRRC8A-/- cells was reduced
compared to HaCaT WT cells. HaCaT LRRC8A-/- and NHEK LRRC8A KO populations showeddecreased RVD compared to the respective WT cells (Trothe et al., 2018).

Kefauver et al., 2018

Patch-clamp recordings of HaCaT WT and LRRC8A-/cells under isotonic and hypotonic conditions

HaCaT WT and LRRC8A-/- cells recorded on the PL,a microfluidic-based APC system
CB(A) LRRC8A WT and KO
currents were recorded using
a voltage ramp protocol from-100 to +100 mV over 200 ms.
Hypotonic solution had anosmolarity of 234 mOsm,
isotonic solution had anosmolarity of 320 mOsm. (B)Time course of the experimentshown in (A). After addition of
hypotonic solution, there wasa large increase in current at
both +100/-100 mV (notshown) followed by block by
100 µM DIDS, only for WT cells.
There was no activation ofLRRC8currents
intheknockout keratinocytes. (C)Average current size inisotonic, hypotonic and in
DIDS for WT (n=11; dark blue)and LRRC8A-/- (n=7; light blue)
HaCaTs. *P<0.05; **P<0.001,paired Student’s t-test.

LRRC8A-/-

WTA

Representative whole-cell current traces of HaCaT WT and LRRC8A-/- example cells in isotonic
external solution (320 mOsmol/kg) and after hypotonic shock, evoked by a voltage-ramp
protocol ranging from -130 to +110 mV, Vhold -50 mV (MPC) or -100 to +100 mV, Vhold -40 mV (PLand SP384).

HaCaT WT and LRRC8A-/- cells recorded on the SP384,
a well-based APC system
AB

D: WT

Patchliner Dynamite8
4 to 8 recording channels(D) Traces from a representative cell in isotonic(left) and hypotonic (right) conditions
recorded using a voltage-step protocol
ranging from -120 to +120 mV, 20 mV steps, for
840 ms (Vhold -40 mV). (E) Current voltage-plot
for an average of 13 WT cells (left) and 5
LRRC8A-/- cells (right) (P<0.05; paired Student’st-test).

BioRender.com

C

BioRender.com

DSyncroPatch 384
384 recording channels(A) Current amplitude elicited using a voltage step to +100 mV (Vhold -40 mV) in isotonic (grey) andhypotonic (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) and LRRC8A-/- (light blue)
keratinocytes is shown as a bar graph, number of cells shown in brackets. The hypotonic-activatedcurrent was significantly larger in WT vs. LRRC8A-/- cells (P<0.05, unpaired Student’s t-test).

EConclusions & Acknowledgements
The Patchliner and SyncroPatch 384 automated patch clamp instruments can be used to record
VRAC channels in keratinocytes which, in combination with WT and KO genotypes, could elucidate
the physiological and pathological role of VRAC diseases, ischemia and even cancer.
This work was supported by the German Federal Ministry of Education and Research through
NatLifE2020 (FKZ031B0089A).