Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis

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.

Keywords: K-Cl cotransporters; KCC3; NKCC1; SPAK; cell volume homeostasis; cerebral edema; neurodegeneration; regulatory volume decrease.

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.

Figure 2

Effect of constitutive KCC3 Thr991/Thr1048 dephosphorylation on KCC3, NKCC1, and NKP activity as a function of flux time. Rb⁺ influx assays in isogenic cell lines harboring inducible expression of either KCC3 wild type (WT) (A) or KCC3 Thr991A/Thr1048A (AA) (B) were performed as described in Methods. Rb⁺ uptake was determined in the following media: total uptake, Cl⁻ alone (Cl), Cl⁻ + ouabain (ClO), Cl⁻ + ouabain + bumetanide (ClOB), S + ouabain + bumetanide (SOB). NKP, NKCC, and KCC represent the Rb⁺ influx through the corresponding transporters. Data for WT (A) were represented in the same scale as for AA (B) cells to show the quantitative difference in Rb⁺ uptake between these two types of cells. Shown are 2 independent experiments, n = 9 individual determinations for WT cells and n = 4 individual determinations from a single experiment for AA cells. Total number of independent experiments, N = 3 for both WT and AA cells. Data represent the mean ± SEM values. See legend to Figure 1 for further details.

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.

Figure 4

Constitutive KCC3 Thr991/Thr1048 dephosphorylation elicits rapid and potent Cl-dependent and Cl-independent K_i loss. K_i 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 K_i via K⁺ efflux through KCC3. Use of the Cl-free medium SOB partially reduces the magnitude of this K_i decrease, indicating that there are both Cl-dependent and Cl-independent components of the K_i decrease stimulated by KCC3 AA induction. Cl-dependent K⁺ loss in induced cells was calculated as the K_i content in SOB—that in ClO, whereas the Cl-independent K⁺ loss was calculated as the difference in K_i 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.

Figure 5

Constitutive KCC3 dephosphorylation elicits rapid and potent cellular K_i loss, which is time-dependent and partially sensitive to DCPIB. K_i content was measured as described in Materials and Methods during induction with doxycycline (1.0 μg/ml) and in the presence and absence of DCPIB (50 μM), an inhibitor of volume regulated Cl⁻ channels (VRACs) and intermediate K⁺ channels. Black squares, Control, i.e., no doxycycline or DCPIB; red circles, doxycycline alone, and green triangles, doxycycline + DCPIB. Time points where there was a significant change were compared vs. the Control. n = 8 individual determinations. Total number of independent identical experiments N = 2. ^*p < 0.001; data represent the mean ± SEM values.

Figure 6

Constitutive KCC3 Thr991/Thr1048 dephosphorylation elicits rapid and potent cellular K_i loss which is time-dependent and partially sensitive to DCPIB. K_i content was measured as described in Materials and Methods during induction with doxycycline (1 μg/ml) and in the presence and absence of DCPIB (50 μM). (A) K_i content in Cl-free medium, sulfamate (S) replacement without (SOB, black squares) and with DCPIB (SOBD, red circles). (B) K_i content in Cl-media ± ouabain (0.1 mM) ± bumetanide (10 μM) without (Cl, ClO and ClOB, black squares, red circles, and green triangles, respectively) and with DCPIB (ClD, ClOD and ClOBD, blue triangles point down, cyan diamonds, and magenta triangles point left, respectively). Mean ± SE, n = 3–6 individual determinations. (A) For SOB vs. SOBD, there was a statistically significant difference between groups as determined by One-Way ANOVA [F_{(8, 21)} = 51.65, p < 0.0005, n = 30], and confirmed by paired t-test, ^*p ≤ 0.01 at 3 and 6 h and ^#p ≤ 0.005 at 9 h. (B) For Cl, ClO, ClOB vs. ClD, ClOD, ClOBD, respectively, there was a statistically significant difference between groups as determined by Kruskal–Wallis One–Way ANOVA [F_{(8, 21)} = 64.42, 77.64, and 45.44, p < 0.0005, n = 30], and confirmed by paired t-test, ^*p < 0.05 at 3 h and ^#p < 0.005 at 6 and 9 h. Data represent the mean ± SEM values. Representative results from 4 similar experiments.

Figure 7

Constitutive KCC3 Thr991/Thr1048 dephosphorylation elicits potent Rb⁺ influx through ouabain-sensitive (NKP) and Cl-dependent (NKCC and KCC) pathways that are time-dependent and partially sensitive to DCPIB. Rb⁺ influx was measured simultaneously with the K_i contents of Figure 6 and as described in Materials and Methods, during induction with doxycycline (1 μg/ml) and ± DCPIB (50 μM). (A–C) Represent NKP, NKCC, and KCC without and with DCPIB (NKPD, NKCCD, and KCCD), respectively. Black squares in the absence and red circles in the presence of DCPIB. n = 3–6 individual determinations. There was a statistically significant difference between groups as determined by One-Way ANOVA for NKP vs. NKPD [F_{(8, 19)} = 26.06], NKCC vs. NKCCD [F_{(8, 21)} = 41.81] and KCC vs. KCCD [F_{(8, 21)} = 368.3], p < 0.0005, n = 30. Data represent the mean ± SEM values. Representative results from 4 similar experiments.

Figure 8

Cl-dependent Rb⁺ influx and K_i content in constitutively dephosphorylated KCC3 cells are DCPIB-sensitive. Lines indicate doxycycline-induced Rb⁺ influx (A) and K_i 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 K_i 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.

Figure 9

Cl-independent K_i loss trigged by constitutive KCC3 Thr991/Thr1048 dephosphorylation is DCPIB-sensitive. Bars indicate total doxycycline-induced K_i loss (T, red), K_i loss through Cl-dependent cotransporters (NKCC + KCC, ClCOT, green), and K⁺ channel-sensitive K_i loss (KCH, blue) when DCPIB, an inhibitor of volume regulated Cl⁻ channels (VRACs) and intermediate K⁺ channels, was applied in the induction (I), preincubation (P), and/or flux (F) stages of the ion flux study. DCPIB inhibits Cl-sensitive K_i loss. Representative results from a typical experiment done in quadruplicates. Total number of similar experiments, N = 4. For T, there was a statistically significant difference between groups as determined by One-Way ANOVA [F_{(7, 24)} = 5.15, p < 0.001, n = 32] or by Kruskal–Wallis One-Way ANOVA [F_{(7, 24)} = 4.46, p < 0.005, n = 32]. There was a statistically significant difference between groups as determined by Kruskal–Wallis One-Way ANOVA [F_{(8, 27)} = 12.01, p < 0.0005, n = 36] for ClCOT but not for KCH [F_{(7, 24)} = 1.41, p > 0.2, n = 32. Data represent the mean ± SEM values. However, there was a statistically significant difference for KCH, ^*p < 0.05 and ^#p < 0.005; and for TD (&p < 0.0005) by paired T-Test for the different conditions shown in the figure and described in the text.

Figure 10

Cl-dependent Rb⁺ influx and K_i loss triggered by constitutive KCC3 Thr991/Thr1048 dephosphorylation is DCPIB-sensitive. Bars indicate Rb⁺ influx through Cl-dependent cotransporters (NKCC + KCC, cyan) and K_i 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 K_i 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.

Figure 11

Constitutive KCC3 Thr991/Thr1048 dephosphorylation reduces acute cell swelling in response to hypotonic osmotic stress. (A) Representative relative change in cell volume after hypotonic swelling in KCC3 WT and KCC3 AA cells (see Materials and Methods for details). Cells were exposed to isotonic HEPES-MEM (310 mOsm/kg H₂O), followed by hypotonic HEPES-MEM (150 mOsm/kg H₂O) for 20 min, and subsequently isotonic HEPES-MEM for 5 min. For drug treatment, cells were pre-incubated with 2 mM furosemide for 15 min prior to the osmotic stress. Furosemide was present throughout the experiment. (B) Summary data of cell volume increase. Inset shows representative traces of relative change in cell water in KCC3 AA cells in presence or absence of furosemide. Data are means ± SE. n = 5, ^*P < 0.05 vs. WT control. P = 0.1 WT + furosemide vs. AA + furosemide. (C) Rate of cell volume change. Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell water volume data (relative change) during the initial swelling response (5–6 min, inset: slopes in red). ^*p < 0.05 vs. WT. (D) Regulatory volume decrease (RVD). Bar graphs represent the rate constants from the slopes (red line) calculated by fitting a linear regression to the cell volume relative change data during 10–24 min of cell volume recovery (inset: slopes in red). Data are means ± SE. n = 5 experiments. ^*p < 0.05 vs. WT.