202 The importance of LRRC8 ion channel in keratinocyte differentiation

Epidermal regeneration relies on a balance between keratinocyte proliferation and differentiation. Both processes are accompanied by cell volume changes. The anion channel LRRC8 mediates regulation upon hypotonic stress in keratinocytes its essential subunit LRRC8A is mainly expressed the basal, transient amplifying population of human epidermis. In vitro differentiation experiments primary confirmed these findings, as upregulated during onset leading to bell-shaped expression pattern. Therefore, we asked whether also involved process changes keratinocytes. order understand role performed different loss-of-function strategies. First, used CRISPR/Cas9 edit HaCaT cells immortalized line NHEK/SV-TERT. contrast cells, NHEK-SV/TERT can differentiate after Ca2+ switch 2D show stratification epidermal equivalents. both lines, found aberrant 3D experiments. Next, siRNA reduce levels keratinocytes, which better simulate vivo This significantly reduced RNA protein ion activity. Surprisingly, knockdown did not interfere with As has short window high initiation, specifically targeted siRNA. Strikingly, indeed leads capability. Our work identifies an molecule differentiation, main function where needed for from