The K‐Cl cotransporter KCC3 is a catalytic switch of cellular K and volume homoestasis

Abstract

The SLC12A cation cotransporters NKCC1 and KCCs play a crucial role in volume homeostasis, with KCC3 as key mediator of regulatory volume decrease. Dephosphorylation of human KCC3 at T991 & T1048 by protein phosphatase 1is triggered by cell swelling, but its physiological effects have not been systematically explored. By creating isogenic human epithelial cell lines with inducible expression of wild type KCC3 (WT) or a constitutively‐active double alanine KCC3 mutant mimicking the dephosphorylated KCC3 (AA), and measuring KCC3 activity with Rb, a K congener, and cell K (Ki) ± transport inhibitors, we gain insight into the role of KCC3 in cell K and volume homeostasis. In isotonic medium, WT is phosphorylated and of low activity, as assessed by phospho‐specific antibodies against T991 & T1048 and Rb flux. In contrast, induction of AA resulted in a 10 fold greater furosemide‐sensitive (FS) KCC3 activity. In induced AA but not WT cells, Ki loss was >; 90 % in Na and ~ 70 % in Na‐free media, time‐ and external K and Rb concentrations‐dependent and could not be prevented by various Cl, K and Ca channel blockers. In contrast to NKCC and KCC, the induced Ki loss was insensitive to F in the preincubation and flux. Addition of F during induction of AA significantly reduced Ki loss in sulfamate with concomitant inhibition of KCC. Thus, a constitutively active KCC3 promotes an equal FS Ki loss and Rb influx, which may impact certain pathologies. Supported by WSU Foundation and NIH R25.