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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.


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Cl-dependent Rb+ influx and Ki content in constitutively dephosphorylated KCC3 cells are DCPIB-sensitive. Lines indicate doxycycline-induced Rb+ influx (A) and Ki content (B), which were assessed in the following media: Cl− + 0.1 mM ouabain (ClO), Cl− + (ouabain + 10 μM bumetanide) (ClOB), S + (ouabain + bumetanide) (SOB) after induction of KCC3 T991A/T1048A (AA) (see Materials and Methods for details). (A) Calculated Rb+ flux through NKCC, black squares and KCC, red circles as the Cl-dependent, ouabain-insensitive, bumetanide-sensitive Rb+ influx, and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx, respectively. Calculation of NKCC in AA cells after induction may result in a negative or positive value depending on the relative magnitude of the Rb+ influx in ClOB with respect to ClO, i.e., CLO − CLOB = + or − NKCC (A); for Ki content (B) ClO, black squares, CLOB, red circles and SOB, green triangles (see Results and Discussion for further details). Flux time, 5 min; n = 4 individual determinations. *p < 0.05 and #p < 0.005 as determined by paired t-test with respect to 0 μM DCPIB; data represent the mean ± SEM values. Representative results from 4 similar experiments.
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Figure 8: Cl-dependent Rb+ influx and Ki content in constitutively dephosphorylated KCC3 cells are DCPIB-sensitive. Lines indicate doxycycline-induced Rb+ influx (A) and Ki content (B), which were assessed in the following media: Cl− + 0.1 mM ouabain (ClO), Cl− + (ouabain + 10 μM bumetanide) (ClOB), S + (ouabain + bumetanide) (SOB) after induction of KCC3 T991A/T1048A (AA) (see Materials and Methods for details). (A) Calculated Rb+ flux through NKCC, black squares and KCC, red circles as the Cl-dependent, ouabain-insensitive, bumetanide-sensitive Rb+ influx, and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx, respectively. Calculation of NKCC in AA cells after induction may result in a negative or positive value depending on the relative magnitude of the Rb+ influx in ClOB with respect to ClO, i.e., CLO − CLOB = + or − NKCC (A); for Ki content (B) ClO, black squares, CLOB, red circles and SOB, green triangles (see Results and Discussion for further details). Flux time, 5 min; n = 4 individual determinations. *p < 0.05 and #p < 0.005 as determined by paired t-test with respect to 0 μM DCPIB; data represent the mean ± SEM values. Representative results from 4 similar experiments.

Mentions: Unexpectedly, DCPIB, a blocker of volume-regulated Cl− channel activity (VRAC, or ICl−swell), and known to inhibit K+ loss through intermediate K+ (IK; KCa3.1) conductance channels (Lauf et al., 2008) caused partial inhibition of K+ loss during induction (Figure 5). Furthermore, DCPIB inhibited KCC3 AA-induced Cl-independent Ki loss (Figure 6) while inhibiting KCC and NKP and activating NKCC (Figure 7). DCPIB, dose-dependently, also increased NKCC and Ki content but inhibited KCC as a function of time when present during the flux (Figure 8).


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 content in constitutively dephosphorylated KCC3 cells are DCPIB-sensitive. Lines indicate doxycycline-induced Rb+ influx (A) and Ki content (B), which were assessed in the following media: Cl− + 0.1 mM ouabain (ClO), Cl− + (ouabain + 10 μM bumetanide) (ClOB), S + (ouabain + bumetanide) (SOB) after induction of KCC3 T991A/T1048A (AA) (see Materials and Methods for details). (A) Calculated Rb+ flux through NKCC, black squares and KCC, red circles as the Cl-dependent, ouabain-insensitive, bumetanide-sensitive Rb+ influx, and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx, respectively. Calculation of NKCC in AA cells after induction may result in a negative or positive value depending on the relative magnitude of the Rb+ influx in ClOB with respect to ClO, i.e., CLO − CLOB = + or − NKCC (A); for Ki content (B) ClO, black squares, CLOB, red circles and SOB, green triangles (see Results and Discussion for further details). Flux time, 5 min; n = 4 individual determinations. *p < 0.05 and #p < 0.005 as determined by paired t-test with respect to 0 μM DCPIB; data represent the mean ± SEM values. Representative results from 4 similar experiments.
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Figure 8: Cl-dependent Rb+ influx and Ki content in constitutively dephosphorylated KCC3 cells are DCPIB-sensitive. Lines indicate doxycycline-induced Rb+ influx (A) and Ki content (B), which were assessed in the following media: Cl− + 0.1 mM ouabain (ClO), Cl− + (ouabain + 10 μM bumetanide) (ClOB), S + (ouabain + bumetanide) (SOB) after induction of KCC3 T991A/T1048A (AA) (see Materials and Methods for details). (A) Calculated Rb+ flux through NKCC, black squares and KCC, red circles as the Cl-dependent, ouabain-insensitive, bumetanide-sensitive Rb+ influx, and Cl-dependent, ouabain and bumetanide-insensitive Rb+ influx, respectively. Calculation of NKCC in AA cells after induction may result in a negative or positive value depending on the relative magnitude of the Rb+ influx in ClOB with respect to ClO, i.e., CLO − CLOB = + or − NKCC (A); for Ki content (B) ClO, black squares, CLOB, red circles and SOB, green triangles (see Results and Discussion for further details). Flux time, 5 min; n = 4 individual determinations. *p < 0.05 and #p < 0.005 as determined by paired t-test with respect to 0 μM DCPIB; data represent the mean ± SEM values. Representative results from 4 similar experiments.
Mentions: Unexpectedly, DCPIB, a blocker of volume-regulated Cl− channel activity (VRAC, or ICl−swell), and known to inhibit K+ loss through intermediate K+ (IK; KCa3.1) conductance channels (Lauf et al., 2008) caused partial inhibition of K+ loss during induction (Figure 5). Furthermore, DCPIB inhibited KCC3 AA-induced Cl-independent Ki loss (Figure 6) while inhibiting KCC and NKP and activating NKCC (Figure 7). DCPIB, dose-dependently, also increased NKCC and Ki content but inhibited KCC as a function of time when present during the flux (Figure 8).

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