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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 elicits rapid and potent Cl-dependent and Cl-independent Ki loss. Ki after flux in 10 mM RbCl with ouabain (ClO); ouabain + bumetanide (ClOB); and in 10 mM RbS with ouabain + bumetanide (SOB) in the indicated cell lines [A (WT), B (AA)] before and after induction with doxycyline. Induction of KCC3 AA results in a large decrease in Ki via K+ efflux through KCC3. Use of the Cl-free medium SOB partially reduces the magnitude of this Ki decrease, indicating that there are both Cl-dependent and Cl-independent components of the Ki decrease stimulated by KCC3 AA induction. Cl-dependent K+ loss in induced cells was calculated as the Ki content in SOB—that in ClO, whereas the Cl-independent K+ loss was calculated as the difference in Ki content in SOB between non-induced and induced cells. Flux time, 5 min; n = 9 individual determinations from a single experiment for WT and n = 4 for AA cells. Total number of independent experiments N = 4 for WT and N = 2 for AA cells. There was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA for Non-induced vs. Induced (ClO, ClOB, and SOB) for WT [F(5, 48) = 21.55] and AA [F(5, 18) = 36.97], p < 0.0005, n = 54 and 24, respectively. These differences were confirmed by paired t-test for (ClOB vs. SOB) in Non-induced and Induced WT, *p < 0.0005, and AA, +p < 0.05 (Non-induced) and *p < 0.0005 (Induced); whereas the comparison between Non-induced vs. Induced AA was *p < 0.0005 for ClO and ClOB, and #p < 0.005 for SOB. Data represent the mean ± SEM values.
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Figure 4: Constitutive KCC3 Thr991/Thr1048 dephosphorylation elicits rapid and potent Cl-dependent and Cl-independent Ki loss. Ki after flux in 10 mM RbCl with ouabain (ClO); ouabain + bumetanide (ClOB); and in 10 mM RbS with ouabain + bumetanide (SOB) in the indicated cell lines [A (WT), B (AA)] before and after induction with doxycyline. Induction of KCC3 AA results in a large decrease in Ki via K+ efflux through KCC3. Use of the Cl-free medium SOB partially reduces the magnitude of this Ki decrease, indicating that there are both Cl-dependent and Cl-independent components of the Ki decrease stimulated by KCC3 AA induction. Cl-dependent K+ loss in induced cells was calculated as the Ki content in SOB—that in ClO, whereas the Cl-independent K+ loss was calculated as the difference in Ki content in SOB between non-induced and induced cells. Flux time, 5 min; n = 9 individual determinations from a single experiment for WT and n = 4 for AA cells. Total number of independent experiments N = 4 for WT and N = 2 for AA cells. There was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA for Non-induced vs. Induced (ClO, ClOB, and SOB) for WT [F(5, 48) = 21.55] and AA [F(5, 18) = 36.97], p < 0.0005, n = 54 and 24, respectively. These differences were confirmed by paired t-test for (ClOB vs. SOB) in Non-induced and Induced WT, *p < 0.0005, and AA, +p < 0.05 (Non-induced) and *p < 0.0005 (Induced); whereas the comparison between Non-induced vs. Induced AA was *p < 0.0005 for ClO and ClOB, and #p < 0.005 for SOB. Data represent the mean ± SEM values.

Mentions: Because intracellular ions and water are determinants of cell volume, any inward ion flux study would be incomplete without considering the fate of the intracellular counter-ion content, in our case Ki. Furthermore, as opposed to the Rb+ influx data shown above, KCC-mediated transport actually results in K+ loss (efflux) and requires Cl−. Figure 4 shows Ki in KCC3 WT (Figure 4A) and KCC3 AA (Figure 4B) cells measured after exposure to Cl− or S media in conditions of NKP inhibition by ouabain, and NKCC inhibition by ouabain + bumetanide, in both non-induced and induced cells. Note that in KCC3 WT cells, there was a 30 % loss of Ki in Cl-free medium (601 ± 5.2 in ClOB vs. 417 ± 10.7 in SOB, i.e., 31.5 ± 1.5 % in non-induced and 609 ± 10.6 in ClOB and 423 ± 15.9, i.e., 30.6 ± 1.8 % in induced WT cells, n = 9 individual determinations for all the conditions).


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 elicits rapid and potent Cl-dependent and Cl-independent Ki loss. Ki after flux in 10 mM RbCl with ouabain (ClO); ouabain + bumetanide (ClOB); and in 10 mM RbS with ouabain + bumetanide (SOB) in the indicated cell lines [A (WT), B (AA)] before and after induction with doxycyline. Induction of KCC3 AA results in a large decrease in Ki via K+ efflux through KCC3. Use of the Cl-free medium SOB partially reduces the magnitude of this Ki decrease, indicating that there are both Cl-dependent and Cl-independent components of the Ki decrease stimulated by KCC3 AA induction. Cl-dependent K+ loss in induced cells was calculated as the Ki content in SOB—that in ClO, whereas the Cl-independent K+ loss was calculated as the difference in Ki content in SOB between non-induced and induced cells. Flux time, 5 min; n = 9 individual determinations from a single experiment for WT and n = 4 for AA cells. Total number of independent experiments N = 4 for WT and N = 2 for AA cells. There was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA for Non-induced vs. Induced (ClO, ClOB, and SOB) for WT [F(5, 48) = 21.55] and AA [F(5, 18) = 36.97], p < 0.0005, n = 54 and 24, respectively. These differences were confirmed by paired t-test for (ClOB vs. SOB) in Non-induced and Induced WT, *p < 0.0005, and AA, +p < 0.05 (Non-induced) and *p < 0.0005 (Induced); whereas the comparison between Non-induced vs. Induced AA was *p < 0.0005 for ClO and ClOB, and #p < 0.005 for SOB. Data represent the mean ± SEM values.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Constitutive KCC3 Thr991/Thr1048 dephosphorylation elicits rapid and potent Cl-dependent and Cl-independent Ki loss. Ki after flux in 10 mM RbCl with ouabain (ClO); ouabain + bumetanide (ClOB); and in 10 mM RbS with ouabain + bumetanide (SOB) in the indicated cell lines [A (WT), B (AA)] before and after induction with doxycyline. Induction of KCC3 AA results in a large decrease in Ki via K+ efflux through KCC3. Use of the Cl-free medium SOB partially reduces the magnitude of this Ki decrease, indicating that there are both Cl-dependent and Cl-independent components of the Ki decrease stimulated by KCC3 AA induction. Cl-dependent K+ loss in induced cells was calculated as the Ki content in SOB—that in ClO, whereas the Cl-independent K+ loss was calculated as the difference in Ki content in SOB between non-induced and induced cells. Flux time, 5 min; n = 9 individual determinations from a single experiment for WT and n = 4 for AA cells. Total number of independent experiments N = 4 for WT and N = 2 for AA cells. There was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA for Non-induced vs. Induced (ClO, ClOB, and SOB) for WT [F(5, 48) = 21.55] and AA [F(5, 18) = 36.97], p < 0.0005, n = 54 and 24, respectively. These differences were confirmed by paired t-test for (ClOB vs. SOB) in Non-induced and Induced WT, *p < 0.0005, and AA, +p < 0.05 (Non-induced) and *p < 0.0005 (Induced); whereas the comparison between Non-induced vs. Induced AA was *p < 0.0005 for ClO and ClOB, and #p < 0.005 for SOB. Data represent the mean ± SEM values.
Mentions: Because intracellular ions and water are determinants of cell volume, any inward ion flux study would be incomplete without considering the fate of the intracellular counter-ion content, in our case Ki. Furthermore, as opposed to the Rb+ influx data shown above, KCC-mediated transport actually results in K+ loss (efflux) and requires Cl−. Figure 4 shows Ki in KCC3 WT (Figure 4A) and KCC3 AA (Figure 4B) cells measured after exposure to Cl− or S media in conditions of NKP inhibition by ouabain, and NKCC inhibition by ouabain + bumetanide, in both non-induced and induced cells. Note that in KCC3 WT cells, there was a 30 % loss of Ki in Cl-free medium (601 ± 5.2 in ClOB vs. 417 ± 10.7 in SOB, i.e., 31.5 ± 1.5 % in non-induced and 609 ± 10.6 in ClOB and 423 ± 15.9, i.e., 30.6 ± 1.8 % in induced WT cells, n = 9 individual determinations for all the conditions).

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