Here we show that experimentally increased O-GlcNAcylation of cellular proteins inhibited the endogenous as well as the ICln-induced IClswell current and prevented RVD in a human renal cell line, while decreases in O-GlcNAcylation augmented the current magnitude. In parallel, increases or decreases in O-GlcNAcylation respectively weakened or stabilized the binding of ICln to the intracellular domain of α-integrin, a process that is essential for the activation of IClswell. Mutation of the putative Yin-O-Yang site at Ser67 rendered the ICln-induced IClswell current unresponsive to O-GlcNAc variations, and the ICln interaction with α-integrin insensitive to O-GlcNAcylation. In addition, exposure of cells to a hypotonic solution reduced the O-GlcNAcylation of cellular proteins. Together, these findings show that O-GlcNAcylation affects RVD by influencing IClswell, and further indicate that hypotonicity may activate IClswell by reducing the O-GlcNAcylation of ICln at Ser67, therefore permitting its binding to α-integrin. We propose that disturbances in the regulation of cellular volume may contribute to disease in settings of chronically elevated O-GlcNAcylation, including diabetic nephropathy. 2020-11-06 Zoltan Szabo University of Szeged Dr. Silvia Dossena ICLN, O-GlcNAcylation 170904-PTE-GLNAC-1.raw raw 677627677 Homo sapiens (Human) cell culture (none) immunoprecipitation Trypsin Carbamidomethyl (C) Oxidation (M) HexNAc (S) HexNAc (T) Oxidation (M) Q Exactive DDA-high res. Qualification - 170904-PTE-GLNAC-1.mgf peak 86727102 MGF 170904-PTE-GLNAC-1 (F001619).mzid result 19998887 mzIdentML Mascot 170904-PTE-GLNAC-1 (F001619).mzid_170904-PTE-GLNAC-1_(F001619).MGF peak 4026982 MGF