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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

Cl-dependent Rb+ influx and Ki loss triggered by constitutive KCC3 Thr991/Thr1048 dephosphorylation is DCPIB-sensitive. Bars indicate Rb+ influx through Cl-dependent cotransporters (NKCC + KCC, cyan) and Ki loss (NKCC + KCC, magenta), when DCPIB was applied in the induction (I), preincubation (P), and/or flux (F) stages of the ion flux study. DCPIB inhibits Cl-dependent Rb+ influx and Ki loss depending on the conditions. Representative results from 4 similar experiments. For Rb+ (NKCC + KCC), there was a statistically significant difference between groups as determined by One-Way ANOVA [F(8, 27) = 48.75, p < 0.0005, n = 36]. For K+ (NKK + KCC), there was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA [F(8, 27) = 20.51, p < 0.0005, n = 36]. Data represent the mean ± SEM values.
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Figure 10: Cl-dependent Rb+ influx and Ki loss triggered by constitutive KCC3 Thr991/Thr1048 dephosphorylation is DCPIB-sensitive. Bars indicate Rb+ influx through Cl-dependent cotransporters (NKCC + KCC, cyan) and Ki loss (NKCC + KCC, magenta), when DCPIB was applied in the induction (I), preincubation (P), and/or flux (F) stages of the ion flux study. DCPIB inhibits Cl-dependent Rb+ influx and Ki loss depending on the conditions. Representative results from 4 similar experiments. For Rb+ (NKCC + KCC), there was a statistically significant difference between groups as determined by One-Way ANOVA [F(8, 27) = 48.75, p < 0.0005, n = 36]. For K+ (NKK + KCC), there was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA [F(8, 27) = 20.51, p < 0.0005, n = 36]. Data represent the mean ± SEM values.

Mentions: To further assess the effect of the VRAC inhibitor on the main transporters studied, DCPIB (50 μM) was applied during the 3 main steps of the Rb+ influx protocol; i.e., during KCC3 AA induction (I), pre-incubation (P), flux (F), and in different combinations (IP, IF, and IPF) (Figure 9, Supplementary Tables 1, 2). Three main components for K+ loss were defined and were calculated as total KCC3 AA-induced K+ loss (T), Cl− cotransporter-dependent K+ loss (ClCOT, i.e., through NKCC + KCC, see Figure 10), and K+ channel-dependent K+ loss (KCH). Figure 9 depicts the total KCC3 AA-induced K+ loss (T) through its two components (ClCOT and KCH) in the various 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)

Cl-dependent Rb+ influx and Ki loss triggered by constitutive KCC3 Thr991/Thr1048 dephosphorylation is DCPIB-sensitive. Bars indicate Rb+ influx through Cl-dependent cotransporters (NKCC + KCC, cyan) and Ki loss (NKCC + KCC, magenta), when DCPIB was applied in the induction (I), preincubation (P), and/or flux (F) stages of the ion flux study. DCPIB inhibits Cl-dependent Rb+ influx and Ki loss depending on the conditions. Representative results from 4 similar experiments. For Rb+ (NKCC + KCC), there was a statistically significant difference between groups as determined by One-Way ANOVA [F(8, 27) = 48.75, p < 0.0005, n = 36]. For K+ (NKK + KCC), there was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA [F(8, 27) = 20.51, p < 0.0005, n = 36]. Data represent the mean ± SEM values.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Cl-dependent Rb+ influx and Ki loss triggered by constitutive KCC3 Thr991/Thr1048 dephosphorylation is DCPIB-sensitive. Bars indicate Rb+ influx through Cl-dependent cotransporters (NKCC + KCC, cyan) and Ki loss (NKCC + KCC, magenta), when DCPIB was applied in the induction (I), preincubation (P), and/or flux (F) stages of the ion flux study. DCPIB inhibits Cl-dependent Rb+ influx and Ki loss depending on the conditions. Representative results from 4 similar experiments. For Rb+ (NKCC + KCC), there was a statistically significant difference between groups as determined by One-Way ANOVA [F(8, 27) = 48.75, p < 0.0005, n = 36]. For K+ (NKK + KCC), there was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA [F(8, 27) = 20.51, p < 0.0005, n = 36]. Data represent the mean ± SEM values.
Mentions: To further assess the effect of the VRAC inhibitor on the main transporters studied, DCPIB (50 μM) was applied during the 3 main steps of the Rb+ influx protocol; i.e., during KCC3 AA induction (I), pre-incubation (P), flux (F), and in different combinations (IP, IF, and IPF) (Figure 9, Supplementary Tables 1, 2). Three main components for K+ loss were defined and were calculated as total KCC3 AA-induced K+ loss (T), Cl− cotransporter-dependent K+ loss (ClCOT, i.e., through NKCC + KCC, see Figure 10), and K+ channel-dependent K+ loss (KCH). Figure 9 depicts the total KCC3 AA-induced K+ loss (T) through its two components (ClCOT and KCH) in the various 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