Hypotonic stress response of human keratinocytes involves LRRC8A as component of volume‐regulated anion channels

Abstract

The barrier function of the human epidermis is constantly challenged by environmental osmotic fluctuations. Hypotonic stress triggers cell swelling, which is counteracted by a compensatory mechanism called regulatory volume decrease (RVD) involving volume‐regulated anion channels (VRACs). Recently, it was discovered that VRACs are composed of LRRC8 heteromers and that LRRC8A functions as the essential VRAC subunit in various mammalian cell types; however, the molecular identity of VRACs in the human epidermis remains to be determined. Here, we investigated the expression of LRRC8A and its role in hypotonic stress response of human keratinocytes. Immunohistological staining showed that LRRC8A is preferentially localized in basal and suprabasal epidermal layers. RNA sequencing revealed that LRRC8A is the most abundant subunit within the LRRC8 gene family in HaCaT cells as well as in primary normal human epidermal keratinocytes (NHEKs). To determine the contribution of LRRC8A to hypotonic stress response, we generated HaCaT‐ and NHEK‐LRRC8A knockout cells by using CRISPR‐Cas9. I− influx assays using halide‐sensitive YFP showed that LRRC8A is crucially important for mediating VRAC activity in HaCaTs and NHEKs. Moreover, cell volume measurements using calcein‐AM dye further revealed that LRRC8A also substantially contributes to RVD. In summary, our study provides new insights into hypotonic stress response and suggests an important role of LRRC8A as VRAC component in human keratinocytes.

The LRRC8 volume‐regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel

“…However, the genes encoding the channels remained elusive and highly controversial until 2014 when two independent groups definitively identified the Leucine‐Rich Repeat Containing 8A‐E (LRRC8A‐E) genes that encode VRACs (Kumar et al, ; Voss et al, ) Results from gene disruption, heterologous expression, and cryo‐EM experiments indicate that VRACs are likely hexameric channels that contain LRRC8A and at least one other LRRC8 subunit (i.e., LRRC8B, LRRC8C, LRRC8D, or LRRC8E) (Strange et al, ). Importantly for the present study, several groups have shown that genetic disruption of the LRRC8A gene abolishes VRAC activity in a variety of different cell types (Bao et al, ; Platt et al, ; Stuhlmann, Planells‐Cases, & Jentsch, ; Trothe et al, ).…”

Profiling Cisplatin Resistance in Head and Neck Cancer: A Critical Role of the VRAC Ion Channel for Chemoresistance

“…LRRC8A-KO cells were prepared using CRISPR/Cas9 tools, as has been described [ 53 ]; however, plasmids were used for expression cassette delivery. Cells were transfected with plasmids (2 µg) using Lipofectamin 2000 according to the manufacturer’s instructions and potentially edited cells selected via Blasticidin treatment (Fisher Scientific GmbH, Schwerte, Germany, 2 µg/µL) for 9 d [ 53 ]. For primer sequences, see Supplementary Table S2 .…”

Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells

“…Since the molecular structure of VRAC was identified, a series of antibodies against different regions of the basic subunits of the family LRRC8 have been developed and effectively used [4,5,9,14,17,18,[23][24][25][26]. Before now, VRAC subunit proteins were detected mostly by immunoblotting of the solubilized whole cells [27][28][29][30][31][32], less often of cell membranes lysates [14,33,34] or by imaging cells that have been permeabilized for staining [35,36]. Furthermore, these data were mostly obtained from cells specifically rich in VRAC such as epithelial cells, astrocytes, vascular smooth muscle cells and transfected cells.…”

“…Surface localization of LRRC8A has been shown in several cases [4,[36][37][38]. Intracellular and plasma membrane localization of GFP-tagged LRRC8A was visualized in living KCP-4 and KB cell lines by immunoimaging [30].…”