The Phosphorylation State of KCC3 Encodes a Potent Molecular Switch of Transporter Activity, Cell Volume, and K + Content

Abstract

Defense of cell volume against excessive shrinkage or swelling is a requirement for multiple essential cellular functions and organismal survival. Cell swelling triggers a homeostatic counter-response. regulatory volume decrease (RVD), resulting in K+ and Cl– efflux (and osmotically-obliged water) via the activation of K+ channels, volume-regulated anion channels (VRACs)), and K+-Cl– cotransporters (KCCs) such as KCC3 (SLC12A6). Here, we characterize a molecular switch of KCC3 activity, cell volume, and intracellular K+ ion content (Ki) encoded in the relative phosphorylation state of two threonines (T991 and T1048) located in the cytoplasmic KCC3 C-terminus. Constitutive dephosphorylation of T991/T1048 stimulates KCC3 activity >25-fold, is accompanied by a complete inhibition (and reversal) of the related bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC1), and results in a rapid (<10 min) and significant (>90%) reduction in Ki via both Cl–-dependent (KCC3 + NKCC1) and Cl–-independent (DCPIB [VRAC inhibitor]-sensitive) pathways. Together, these effects render cells less prone to swelling in even extreme hypotonic stress. We speculate that stimulating KCC3 by antagonizing T991/T1048 phosphorylation might be a novel strategy of countering pathological cell swelling, as seen in cerebral edema, by “re-setting” the cellular osmostat.