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.
Highlights
Regulation of cell volume is critical for multiple essential cellular functions and organismal survival
KCC3 expression and localization was similar across multiple clonal cell lines of both KCC3 WT and KCC3a Thr991A/Thr1048A (KCC3 AA)
Consistent with previous reports (Rinehart et al, 2009; De Los Heros et al, 2014), KCC3 in isotonic conditions was phosphorylated at both Thr991 and Thr1048 (Supplementary Figure 1), as revealed using phosphospecific antibodies raised against these residues (De Los Heros et al, 2014)
Summary
Regulation of cell volume is critical for multiple essential cellular functions and organismal survival. A KCC3 phospho-switch of transporter function respectively (Kregenow, 1971, 1981; Hoffmann and Dunham, 1995; Lauf and Adragna, 2000, 2012; Hoffmann et al, 2009) These mechanisms are highly regulated by signaling molecules that sense changes in intracellular ionic content and/or cell volume, and transduce these signals to the cell membrane to modulate the transport of ions and/or organic osmolytes via the stimulation or inhibition of ion transporters, pumps, and channels (Hoffmann and Dunham, 1995). The four KCC isoforms (KCC1-4) utilize energetically favorable, outwardlydirected K+ gradients to drive the extrusion of Cl− across the plasma membrane As such, they serve as important determinants of both intracellular K+ and Cl− content, which are important for cell volume regulation and other essential functions depending on cell type (e.g., epithelial transport and neuronal excitability) and KCC isoform (Lauf and Adragna, 2012). The physiological importance of the swelling-activated KCCs, and in particular KCC3 (SLC12A6), is clearly illustrated by the phenotypes that result from the knockout of the genes encoding these molecules in mouse (Boettger et al, 2002; Delpire and Mount, 2002), and their mutations in humans (Howard et al, 2002)
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