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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

Rb+ influx via KCC3, NKCC1, and NKP in Na+ and N-methyl-D-glucamine (MDG) medium. Rb+ influx through the Na+/K+ pump (NKP), NKCC and KCC (red, green, and blue bars, respectively) was calculated as ouabain sensitive; Cl-dependent, bumetanide-sensitive; and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx, respectively. KCC3 AA but not KCC3 WT (results not shown) induction causes a potent increase in flux through KCC3, which is accompanied by inhibition of NKCC and stimulation of NKP. The effects of KCC3 AA induction on NKCC and NKP are abolished when extracellular Na+ is replaced with NMDG. n = 6 individual determinations from a single experiment, total number of experiments N = 2. #p < 0.05; *p < 0.005, n = 12, data represent the mean ± SEM values. Two-sample t-test was employed to determine the statistical significance of the differences between Na+ and MDG, as indicated.
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Figure 3: Rb+ influx via KCC3, NKCC1, and NKP in Na+ and N-methyl-D-glucamine (MDG) medium. Rb+ influx through the Na+/K+ pump (NKP), NKCC and KCC (red, green, and blue bars, respectively) was calculated as ouabain sensitive; Cl-dependent, bumetanide-sensitive; and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx, respectively. KCC3 AA but not KCC3 WT (results not shown) induction causes a potent increase in flux through KCC3, which is accompanied by inhibition of NKCC and stimulation of NKP. The effects of KCC3 AA induction on NKCC and NKP are abolished when extracellular Na+ is replaced with NMDG. n = 6 individual determinations from a single experiment, total number of experiments N = 2. #p < 0.05; *p < 0.005, n = 12, data represent the mean ± SEM values. Two-sample t-test was employed to determine the statistical significance of the differences between Na+ and MDG, as indicated.

Mentions: Analysis of the ouabain-sensitive Rb+ uptake component revealed induction of KCC3 AA triggered a ~3–4 fold increase in NKP activation in 5 min (Figure 2B) when compared with NKP activity upon induction of KCC3 WT (Figure 2A, KCC3 WT 23.2 ± 1.0 and KCC3 AA 87.7 ± 12.9 nmol/mg protein − 5 min), consistent with the known 3–4 fold stimulation of NKP flux to its Vmax by intracellular Na+ and extracellular K+ site saturation (reviewed in Lauf and Adragna, 2012). This became further evident when extracellular Na+ replacement by NMDG eliminated NKP activity (Figure 3). No changes were observed in Ki content between non-induced and induced KCC3 WT cells under the same conditions for KCC3 AA cells (data not shown). These data show that prevention of KCC3 Thr991/Thr1048 inhibitory phosphorylation results in constitutive KCC3a activity that is accompanied by concurrent NKCC1 reversal (i.e., outward Rb+ efflux through NKCC1) and NKP stimulation.


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)

Rb+ influx via KCC3, NKCC1, and NKP in Na+ and N-methyl-D-glucamine (MDG) medium. Rb+ influx through the Na+/K+ pump (NKP), NKCC and KCC (red, green, and blue bars, respectively) was calculated as ouabain sensitive; Cl-dependent, bumetanide-sensitive; and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx, respectively. KCC3 AA but not KCC3 WT (results not shown) induction causes a potent increase in flux through KCC3, which is accompanied by inhibition of NKCC and stimulation of NKP. The effects of KCC3 AA induction on NKCC and NKP are abolished when extracellular Na+ is replaced with NMDG. n = 6 individual determinations from a single experiment, total number of experiments N = 2. #p < 0.05; *p < 0.005, n = 12, data represent the mean ± SEM values. Two-sample t-test was employed to determine the statistical significance of the differences between Na+ and MDG, as indicated.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496573&req=5

Figure 3: Rb+ influx via KCC3, NKCC1, and NKP in Na+ and N-methyl-D-glucamine (MDG) medium. Rb+ influx through the Na+/K+ pump (NKP), NKCC and KCC (red, green, and blue bars, respectively) was calculated as ouabain sensitive; Cl-dependent, bumetanide-sensitive; and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx, respectively. KCC3 AA but not KCC3 WT (results not shown) induction causes a potent increase in flux through KCC3, which is accompanied by inhibition of NKCC and stimulation of NKP. The effects of KCC3 AA induction on NKCC and NKP are abolished when extracellular Na+ is replaced with NMDG. n = 6 individual determinations from a single experiment, total number of experiments N = 2. #p < 0.05; *p < 0.005, n = 12, data represent the mean ± SEM values. Two-sample t-test was employed to determine the statistical significance of the differences between Na+ and MDG, as indicated.
Mentions: Analysis of the ouabain-sensitive Rb+ uptake component revealed induction of KCC3 AA triggered a ~3–4 fold increase in NKP activation in 5 min (Figure 2B) when compared with NKP activity upon induction of KCC3 WT (Figure 2A, KCC3 WT 23.2 ± 1.0 and KCC3 AA 87.7 ± 12.9 nmol/mg protein − 5 min), consistent with the known 3–4 fold stimulation of NKP flux to its Vmax by intracellular Na+ and extracellular K+ site saturation (reviewed in Lauf and Adragna, 2012). This became further evident when extracellular Na+ replacement by NMDG eliminated NKP activity (Figure 3). No changes were observed in Ki content between non-induced and induced KCC3 WT cells under the same conditions for KCC3 AA cells (data not shown). These data show that prevention of KCC3 Thr991/Thr1048 inhibitory phosphorylation results in constitutive KCC3a activity that is accompanied by concurrent NKCC1 reversal (i.e., outward Rb+ efflux through NKCC1) and NKP stimulation.

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