title: Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis.
abstract: The defense of cell volume against excessive shrinkage or swelling is a requirement for cell function and organismal survival. Cell swelling triggers a coordinated homeostatic response termed regulatory volume decrease (RVD), resulting in K(+) and Cl(-) efflux via activation of K(+) channels, volume-regulated anion channels (VRACs), and the K(+)-Cl(-) cotransporters, including KCC3. Here, we show genetic alanine (Ala) substitution at threonines (Thr) 991 and 1048 in the KCC3a isoform carboxyl-terminus, preventing inhibitory phosphorylation at these sites, not only significantly up-regulates KCC3a activity up to 25-fold in normally inhibitory isotonic conditions, but is also accompanied by reversal of activity of the related bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter isoform 1 (NKCC1). This results in a rapid (<10 min) and significant (>90%) reduction in intracellular K(+) content (Ki) via both Cl-dependent (KCC3a + NKCC1) and Cl-independent [DCPIB (VRAC inhibitor)-sensitive] pathways, which collectively renders cells less prone to acute swelling in hypotonic osmotic stress. Together, these data demonstrate the phosphorylation state of Thr991/Thr1048 in KCC3a encodes a potent switch of transporter activity, Ki homeostasis, and cell volume regulation, and reveal novel observations into the functional interaction among ion transport molecules involved in RVD.
authors.affiliation: Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University Dayton, OH, USA.
authors.forename: Norma C
authors.initials: NC
authors.lastname: Adragna
authors.affiliation: Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University Dayton, OH, USA.
authors.forename: Nagendra B
authors.initials: NB
authors.lastname: Ravilla
authors.affiliation: Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University Dayton, OH, USA ; Department of Pathology, Boonshoft School of Medicine, Wright State University Dayton, OH, USA.
authors.forename: Peter K
authors.initials: PK
authors.lastname: Lauf
authors.affiliation: Department of Neurology, University of Pittsburgh Pittsburgh, PA, USA.
authors.forename: Gulnaz
authors.initials: G
authors.lastname: Begum
authors.affiliation: Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Harvard University Boston, MA, USA.
authors.forename: Arjun R
authors.initials: AR
authors.lastname: Khanna
authors.affiliation: Department of Neurology, University of Pittsburgh Pittsburgh, PA, USA ; Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center Pittsburgh, PA, USA.
authors.forename: Dandan
authors.initials: D
authors.lastname: Sun
authors.affiliation: Department of Neurosurgery, Boston Children's Hospital and Harvard Medical School, Harvard University Boston, MA, USA ; Manton Center for Orphan Disease Research, Children's Hospital Boston, Harvard University Boston, MA, USA.
authors.forename: Kristopher T
authors.initials: KT
authors.lastname: Kahle
keywords: K-Cl cotransporters; KCC3; NKCC1; SPAK; cell volume homeostasis; cerebral edema; neurodegeneration; regulatory volume decrease