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Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis.

Adragna NC, Ravilla NB, Lauf PK, Begum G, Khanna AR, Sun D, Kahle KT - Front Cell Neurosci (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University Dayton, OH, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Constitutive KCC3 Thr991/Thr1048 dephosphorylation reduces acute cell swelling in response to hypotonic osmotic stress. (A) Representative relative change in cell volume after hypotonic swelling in KCC3 WT and KCC3 AA cells (see Materials and Methods for details). Cells were exposed to isotonic HEPES-MEM (310 mOsm/kg H2O), followed by hypotonic HEPES-MEM (150 mOsm/kg H2O) for 20 min, and subsequently isotonic HEPES-MEM for 5 min. For drug treatment, cells were pre-incubated with 2 mM furosemide for 15 min prior to the osmotic stress. Furosemide was present throughout the experiment. (B) Summary data of cell volume increase. Inset shows representative traces of relative change in cell water in KCC3 AA cells in presence or absence of furosemide. Data are means ± SE. n = 5, *P < 0.05 vs. WT control. P = 0.1 WT + furosemide vs. AA + furosemide. (C) Rate of cell volume change. Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell water volume data (relative change) during the initial swelling response (5–6 min, inset: slopes in red). *p < 0.05 vs. WT. (D) Regulatory volume decrease (RVD). Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell volume relative change data during 10–24 min of cell volume recovery (inset: slopes in red). Data are means ± SE. n = 5 experiments. *p < 0.05 vs. WT.
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Figure 11: Constitutive KCC3 Thr991/Thr1048 dephosphorylation reduces acute cell swelling in response to hypotonic osmotic stress. (A) Representative relative change in cell volume after hypotonic swelling in KCC3 WT and KCC3 AA cells (see Materials and Methods for details). Cells were exposed to isotonic HEPES-MEM (310 mOsm/kg H2O), followed by hypotonic HEPES-MEM (150 mOsm/kg H2O) for 20 min, and subsequently isotonic HEPES-MEM for 5 min. For drug treatment, cells were pre-incubated with 2 mM furosemide for 15 min prior to the osmotic stress. Furosemide was present throughout the experiment. (B) Summary data of cell volume increase. Inset shows representative traces of relative change in cell water in KCC3 AA cells in presence or absence of furosemide. Data are means ± SE. n = 5, *P < 0.05 vs. WT control. P = 0.1 WT + furosemide vs. AA + furosemide. (C) Rate of cell volume change. Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell water volume data (relative change) during the initial swelling response (5–6 min, inset: slopes in red). *p < 0.05 vs. WT. (D) Regulatory volume decrease (RVD). Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell volume relative change data during 10–24 min of cell volume recovery (inset: slopes in red). Data are means ± SE. n = 5 experiments. *p < 0.05 vs. WT.

Mentions: Given the potent Cl-dependent and Cl-independent Ki loss induced by KCC3 AA, we speculated these cells might exhibit different swelling properties compared to those expressing KCC3 WT. We therefore examined cell volume changes and RVD in response to hypotonic challenge in KCC3 WT and KCC3 AA cells (see Materials and Methods for details). At baseline, in isotonic conditions, both cell lines exhibited similar cell volumes (Figure 11A). However, hypotonic conditions [HEPES-MEM (150 mOsm/kg H2O)] elicited a ~3.2 ± 0.5-fold increase in cell volume (peak values) in KCC3 WT cells, compared to significantly less swelling in KCC3 AA cells [1.2 ± 0.3-fold increase in cell volume (peak values)] (Figures 11A,B). Two phases of cell volume changes were further analyzed in Figures 11C,D. First, KCC3 WT cells showed a sharp swelling increase upon hypotonic challenge (at a rate of 1.8 ± 0.3 % cell volume/min); in contrast, KCC3 AA cells exhibited a significantly slower rate (1.05 ± 0.2 % cell volume/min). Secondly, KCC3 WT cells triggered RVD at a rate of 0.13 ± 0.01 % cell volume/min (Figure 11D) and recovered to ~50 ± 0.2 % of their original volume in ~20 min; in contrast, KCC3 AA cells exhibited a slower RVD rate (0.04 ± 0.007 % cell volume/min) (Figure 11D). These data suggest that constitutive KCC3 Thr991/Thr1048 dephosphorylation renders cells less prone to acute swelling in hypotonic osmotic stress.


Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis.

Adragna NC, Ravilla NB, Lauf PK, Begum G, Khanna AR, Sun D, Kahle KT - Front Cell Neurosci (2015)

Constitutive KCC3 Thr991/Thr1048 dephosphorylation reduces acute cell swelling in response to hypotonic osmotic stress. (A) Representative relative change in cell volume after hypotonic swelling in KCC3 WT and KCC3 AA cells (see Materials and Methods for details). Cells were exposed to isotonic HEPES-MEM (310 mOsm/kg H2O), followed by hypotonic HEPES-MEM (150 mOsm/kg H2O) for 20 min, and subsequently isotonic HEPES-MEM for 5 min. For drug treatment, cells were pre-incubated with 2 mM furosemide for 15 min prior to the osmotic stress. Furosemide was present throughout the experiment. (B) Summary data of cell volume increase. Inset shows representative traces of relative change in cell water in KCC3 AA cells in presence or absence of furosemide. Data are means ± SE. n = 5, *P < 0.05 vs. WT control. P = 0.1 WT + furosemide vs. AA + furosemide. (C) Rate of cell volume change. Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell water volume data (relative change) during the initial swelling response (5–6 min, inset: slopes in red). *p < 0.05 vs. WT. (D) Regulatory volume decrease (RVD). Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell volume relative change data during 10–24 min of cell volume recovery (inset: slopes in red). Data are means ± SE. n = 5 experiments. *p < 0.05 vs. WT.
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Figure 11: Constitutive KCC3 Thr991/Thr1048 dephosphorylation reduces acute cell swelling in response to hypotonic osmotic stress. (A) Representative relative change in cell volume after hypotonic swelling in KCC3 WT and KCC3 AA cells (see Materials and Methods for details). Cells were exposed to isotonic HEPES-MEM (310 mOsm/kg H2O), followed by hypotonic HEPES-MEM (150 mOsm/kg H2O) for 20 min, and subsequently isotonic HEPES-MEM for 5 min. For drug treatment, cells were pre-incubated with 2 mM furosemide for 15 min prior to the osmotic stress. Furosemide was present throughout the experiment. (B) Summary data of cell volume increase. Inset shows representative traces of relative change in cell water in KCC3 AA cells in presence or absence of furosemide. Data are means ± SE. n = 5, *P < 0.05 vs. WT control. P = 0.1 WT + furosemide vs. AA + furosemide. (C) Rate of cell volume change. Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell water volume data (relative change) during the initial swelling response (5–6 min, inset: slopes in red). *p < 0.05 vs. WT. (D) Regulatory volume decrease (RVD). Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell volume relative change data during 10–24 min of cell volume recovery (inset: slopes in red). Data are means ± SE. n = 5 experiments. *p < 0.05 vs. WT.
Mentions: Given the potent Cl-dependent and Cl-independent Ki loss induced by KCC3 AA, we speculated these cells might exhibit different swelling properties compared to those expressing KCC3 WT. We therefore examined cell volume changes and RVD in response to hypotonic challenge in KCC3 WT and KCC3 AA cells (see Materials and Methods for details). At baseline, in isotonic conditions, both cell lines exhibited similar cell volumes (Figure 11A). However, hypotonic conditions [HEPES-MEM (150 mOsm/kg H2O)] elicited a ~3.2 ± 0.5-fold increase in cell volume (peak values) in KCC3 WT cells, compared to significantly less swelling in KCC3 AA cells [1.2 ± 0.3-fold increase in cell volume (peak values)] (Figures 11A,B). Two phases of cell volume changes were further analyzed in Figures 11C,D. First, KCC3 WT cells showed a sharp swelling increase upon hypotonic challenge (at a rate of 1.8 ± 0.3 % cell volume/min); in contrast, KCC3 AA cells exhibited a significantly slower rate (1.05 ± 0.2 % cell volume/min). Secondly, KCC3 WT cells triggered RVD at a rate of 0.13 ± 0.01 % cell volume/min (Figure 11D) and recovered to ~50 ± 0.2 % of their original volume in ~20 min; in contrast, KCC3 AA cells exhibited a slower RVD rate (0.04 ± 0.007 % cell volume/min) (Figure 11D). These data suggest that constitutive KCC3 Thr991/Thr1048 dephosphorylation renders cells less prone to acute swelling in hypotonic osmotic stress.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University Dayton, OH, USA.

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.

No MeSH data available.


Related in: MedlinePlus