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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 stimulates KCC3 activity and is accompanied by a reversal of NKCC1 activity. Rb+ influx assays in isogenic cell lines harboring doxycycline-inducible expression of either KCC3 wild type (WT) or non-phosphorylatable KCC3 Thr991A/Thr1048A (AA) were performed as described in Methods. (A) Rb+ influx was assessed in the following media: Cl− + 0.1 mM ouabain (ClO, red), Cl− + (ouabain + 10 μM bumetanide) (ClOB, green), S + (ouabain + bumetanide) (SOB, blue) after induction of the indicated proteins (see Methods for details). (B) Calculated Rb+ flux through NKCC and KCC as the Cl-dependent, ouabain-insensitive, bumetanide-sensitive Rb+ influx (ClO - ClOB), and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx (ClOB - SOB), respectively. Calculation of NKCC in AA cells after induction resulted in a negative value due to a larger Rb+ influx in ClOB than in ClO, i.e., CLO – CLOB = -NKCC (see Results and Discussion for further details). Flux time, 5 min; n = 9 individual determinations for WT cells and n = 4 individual determinations for AA cells. Total number of independent experiments N = 4 for WT and N = 5 for AA cells. Results were similar among different cell clones for both WT and AA. *p < 0.005; #p < 0.0005; data represent the mean ± SEM values. Two-sample t-test was employed to determine the statistical significance of the differences between WT and AA, as indicated.
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Figure 1: Constitutive KCC3 Thr991/Thr1048 dephosphorylation stimulates KCC3 activity and is accompanied by a reversal of NKCC1 activity. Rb+ influx assays in isogenic cell lines harboring doxycycline-inducible expression of either KCC3 wild type (WT) or non-phosphorylatable KCC3 Thr991A/Thr1048A (AA) were performed as described in Methods. (A) Rb+ influx was assessed in the following media: Cl− + 0.1 mM ouabain (ClO, red), Cl− + (ouabain + 10 μM bumetanide) (ClOB, green), S + (ouabain + bumetanide) (SOB, blue) after induction of the indicated proteins (see Methods for details). (B) Calculated Rb+ flux through NKCC and KCC as the Cl-dependent, ouabain-insensitive, bumetanide-sensitive Rb+ influx (ClO - ClOB), and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx (ClOB - SOB), respectively. Calculation of NKCC in AA cells after induction resulted in a negative value due to a larger Rb+ influx in ClOB than in ClO, i.e., CLO – CLOB = -NKCC (see Results and Discussion for further details). Flux time, 5 min; n = 9 individual determinations for WT cells and n = 4 individual determinations for AA cells. Total number of independent experiments N = 4 for WT and N = 5 for AA cells. Results were similar among different cell clones for both WT and AA. *p < 0.005; #p < 0.0005; data represent the mean ± SEM values. Two-sample t-test was employed to determine the statistical significance of the differences between WT and AA, as indicated.

Mentions: Induction of KCC3 WT in isotonic conditions did not elicit a significant KCC-mediated Rb+ influx. For instance, in control cells (i.e., cells not induced with doxycycline) the Rb+ influx was 10.6 ± 1.8 nmol/mg protein × 5 min, whereas in cells induced with doxycycline (shown in Figure 1B), it was 12.4 ± 0.9 nmol/mg protein × 5 min, n = 12 individual determinations from 4 independent experiments, consistent with the presence of inhibitory phosphorylation of KCC3 at Thr991 and Thr1048 (Supplementary Figure 1). Likewise, in KCC3 WT cells, NKCC was 30.2 ± 7.4 in non-induced cells and 29.7 ± 6.0 nmol/mg protein × 5 min in induced cells (shown in Figure 1B), n = 12 individual determinations from 4 independent experiments.


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 stimulates KCC3 activity and is accompanied by a reversal of NKCC1 activity. Rb+ influx assays in isogenic cell lines harboring doxycycline-inducible expression of either KCC3 wild type (WT) or non-phosphorylatable KCC3 Thr991A/Thr1048A (AA) were performed as described in Methods. (A) Rb+ influx was assessed in the following media: Cl− + 0.1 mM ouabain (ClO, red), Cl− + (ouabain + 10 μM bumetanide) (ClOB, green), S + (ouabain + bumetanide) (SOB, blue) after induction of the indicated proteins (see Methods for details). (B) Calculated Rb+ flux through NKCC and KCC as the Cl-dependent, ouabain-insensitive, bumetanide-sensitive Rb+ influx (ClO - ClOB), and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx (ClOB - SOB), respectively. Calculation of NKCC in AA cells after induction resulted in a negative value due to a larger Rb+ influx in ClOB than in ClO, i.e., CLO – CLOB = -NKCC (see Results and Discussion for further details). Flux time, 5 min; n = 9 individual determinations for WT cells and n = 4 individual determinations for AA cells. Total number of independent experiments N = 4 for WT and N = 5 for AA cells. Results were similar among different cell clones for both WT and AA. *p < 0.005; #p < 0.0005; data represent the mean ± SEM values. Two-sample t-test was employed to determine the statistical significance of the differences between WT and AA, as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496573&req=5

Figure 1: Constitutive KCC3 Thr991/Thr1048 dephosphorylation stimulates KCC3 activity and is accompanied by a reversal of NKCC1 activity. Rb+ influx assays in isogenic cell lines harboring doxycycline-inducible expression of either KCC3 wild type (WT) or non-phosphorylatable KCC3 Thr991A/Thr1048A (AA) were performed as described in Methods. (A) Rb+ influx was assessed in the following media: Cl− + 0.1 mM ouabain (ClO, red), Cl− + (ouabain + 10 μM bumetanide) (ClOB, green), S + (ouabain + bumetanide) (SOB, blue) after induction of the indicated proteins (see Methods for details). (B) Calculated Rb+ flux through NKCC and KCC as the Cl-dependent, ouabain-insensitive, bumetanide-sensitive Rb+ influx (ClO - ClOB), and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx (ClOB - SOB), respectively. Calculation of NKCC in AA cells after induction resulted in a negative value due to a larger Rb+ influx in ClOB than in ClO, i.e., CLO – CLOB = -NKCC (see Results and Discussion for further details). Flux time, 5 min; n = 9 individual determinations for WT cells and n = 4 individual determinations for AA cells. Total number of independent experiments N = 4 for WT and N = 5 for AA cells. Results were similar among different cell clones for both WT and AA. *p < 0.005; #p < 0.0005; data represent the mean ± SEM values. Two-sample t-test was employed to determine the statistical significance of the differences between WT and AA, as indicated.
Mentions: Induction of KCC3 WT in isotonic conditions did not elicit a significant KCC-mediated Rb+ influx. For instance, in control cells (i.e., cells not induced with doxycycline) the Rb+ influx was 10.6 ± 1.8 nmol/mg protein × 5 min, whereas in cells induced with doxycycline (shown in Figure 1B), it was 12.4 ± 0.9 nmol/mg protein × 5 min, n = 12 individual determinations from 4 independent experiments, consistent with the presence of inhibitory phosphorylation of KCC3 at Thr991 and Thr1048 (Supplementary Figure 1). Likewise, in KCC3 WT cells, NKCC was 30.2 ± 7.4 in non-induced cells and 29.7 ± 6.0 nmol/mg protein × 5 min in induced cells (shown in Figure 1B), n = 12 individual determinations from 4 independent experiments.

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