Role of TASK2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules

Abstract

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.

Figure 1.

Lac Z expression in renal cells. Kidney sections or primary cultures from TASK2 −/− mice were incubated with X-gal overnight at 37°C. (A) X-gal staining in cortical kidney sections (G, glomerulus; PCT, proximal convoluted tubules; DCT, distal convoluted tubules; CCT, cortical collecting tubules); (B) in primary cultures of proximal cells; (C) in primary cultures of distal cells; and (D) in primary culture of collecting cells.

Figure 2.

Effect of external pH variations on K⁺ currents in cultured PCT cells from TASK2 +/+ and TASK2 −/− mice. Under each experimental condition, the circled values represent the osmotic pressure in the pipette, and the values outside the circle represent the osmotic pressure in the extracellular bath solutions. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. Whole-cell currents were recorded with a 350-mosmol/kg H₂O solution in the extracellular bath containing 5 mM of K⁺, and a 290-mosmol/kg H₂O solution in the pipette containing 125 mM of K⁺. (A and D) Whole-cell currents recorded at a pH of 6.0. (B and E) Whole-cell currents recorded at a pH of 7.4. (C and F) Whole-cell currents recorded at a pH of 8.6. (G and H) Average current-voltage relationships measured at 200 ms after onset of pulse, obtained from same cells at rest. Values are means ± SEM of (n) different cells from six different mice.

Figure 3.

Effect of different K⁺ channel inhibitors and relation between K⁺ currents and pH on PCT cells from TASK2 +/+ mice. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. Whole-cell currents were recorded with solutions containing 125 mM of K⁺ in the pipette and 5 mM of K⁺ in the extracellular bath. (A) Histogram of current values at 100 mV in the presence of different K⁺ channel effectors on K⁺ currents in PCT cells from TASK2 +/+ mice. Whole-cell currents were recorded 3 min after the perfusion of an extracellular solution containing 0.5 mM quinidine, 1 mM TEA, 10 nM CTX, 10 nM apamin, or 10 μM clofilium. Values are measured 200 ms after the onset of pulse at 100 mV. Each value is mean ± SEM of 15 cells obtained from five monolayers. (B) Relation between external pH and K⁺ currents. Whole-cell currents were recorded 3 min after perfusion of an extracellular solution at different pH. Values are measured at 200 ms after the onset of pulse at −40 and +80 mV. Each value is mean ± SEM of eight cells obtained from four monolayers.

Figure 4.

Characteristics of swelling-activated whole-cell K⁺ currents in primary cultures of PCT cells from TASK2 +/+ mice. Under each experimental condition, the circled values represent the osmotic pressure in the pipette, and the values outside the circle represent the osmotic pressure in the extracellular bath solutions. Whole-cell currents were recorded with solutions containing 125 mM of K⁺ in the pipette and 5 mM of K⁺ in extracellular bath. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. (A) Whole-cell currents recorded in control conditions (350 mosmol/kg H₂O in extracellular bath) after the mechanical rupture of membrane. (B) Whole-cell currents recorded 4–5 min after extracellular perfusion of a 290-mosmol/kg H₂O hypotonic solution. (C) Effect of a hypertonic solution (350 mosmol/kg H₂O) on swelling-induced whole-cell K⁺ currents. (D) Average current-voltage relationships measured 200 ms after onset of pulse, obtained from same cell at rest. Values are means ± SEM of 20 different cells from five different monolayers.

Figure 5.

(A) Histogram of percent inhibition of different K⁺ channel inhibitors on swelling-activated K⁺ currents recorded in PCT cells from TASK2 +/+ mice. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. Whole-cell currents were recorded 4–5 min after extracellular perfusion of a 290-mosmol/kg H₂O hypotonic solution in the presence of 10 μM clofilium, 0.5 mM quinidine, 1 mM TEA, 10 nM CTX, and 10 μM 293B. Values measured 200 ms after onset of pulse at 100 mV are converted to percent inhibition. Each value is mean ± SEM of (n) cells obtained from six monolayers. (B) Effect of extracellular pH on the development of swelling-activated K⁺ currents in cultured PCT cells from TASK2 +/+ mice. Under each experimental condition, the circled values represent the osmotic pressure in the pipette and the values outside the circle represent the osmotic pressure in the extracellular bath solutions. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. (Ba, Bb, and Bc) Whole-cell currents were recorded 4–5 min after extracellular perfusion of a 30% hypotonic solution at pH 6.0, 7.4, and 8.6, respectively, in the presence of 5 mM EGTA and 5 mM Mg-ATP in the pipette solution. (C) Corresponding average current-voltage relationships measured 200 ms after onset of pulse, obtained from same cell at rest and during hypotonic stimulation at pH 6.0, 7.4, and 8.6. Values are means ± SEM of (n) cells obtained from five monolayers.

Figure 6.

Characteristics of swelling-activated whole-cell K⁺ currents in primary cultures of PCT cells from TASK2 −/− mice. Under each experimental condition, the circled values represent the osmotic pressure in the pipette, and the values outside the circle represent the osmotic pressure in the extracellular bath solutions. Whole-cell currents were recorded in solutions containing 125 mM of K⁺ in the pipette and 5 mM of K⁺ in the extracellular bath. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. (A) Whole-cell currents recorded in control conditions (350 mosmol/kg H₂O in the extracellular bath) after the mechanical rupture of membrane. (B) Whole-cell currents recorded 4–5 min after extracellular perfusion of a 290-mosmol/kg H₂O hypotonic solution. (C) Average current-voltage relationships measured at 200 ms after onset of pulse, obtained from same cell at rest. Values are means ± SEM of 20 cells from five different monolayers.

Figure 7.

Characteristics of swelling-activated whole-cell Cl⁻ currents in primary cultures of PCT cells from TASK2 −/− mice. Under each experimental condition, the circled values represent the osmotic pressure in the pipette, and the values outside the circle represent the osmotic pressure in the extracellular bath solutions. Whole-cell currents were recorded in solutions containing 140 mM NMDG-Cl in the pipette and in the extracellular bath. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. (A) Whole-cell currents recorded in control conditions (350 mosmol/kg H₂O in the extracellular bath) after mechanical rupture of membrane. (B) Whole-cell currents recorded 4–5 min after extracellular perfusion of a 290-mosmol/kg H₂O hypotonic solution. (C) Effects of a hypertonic solution (350 mosmol/kg H₂O) on swelling-induced whole-cell Cl⁻ currents. (D) Average current-voltage relationships measured at 9 ms after onset of pulse. Each value is mean ± SEM of five cells obtained from three monolayers.

Figure 8.

RVD in cultured PCT cells from TASK2 +/+ and TASK2 −/− mice. After loading with Fura2, cultures were rinsed in 300 mosmol/kg H₂O NaCl solution, and fluorescence of Fura2 was measured at 360 nm for a control period of 1 min. Then, a hypotonic shock was induced by perfusing a 200-mosmol/kg H₂O NaCl solution. Images were recorded every 5 s. After analysis, relative volume change in percentage of initial volume was plotted against time as explained in materials and methods. (A) Effect of a hypotonic shock on primary cultured cells. Measurements were performed on six monolayers (25 random cells each) from TASK2 −/− and five monolayers (25 random cell each) from TASK2 +/+. (B) Effect of external Ca²⁺ on RVD in PCT cells from TASK2 +/+ mice. Measurements were performed on four monolayers (25 random cells each) from TASK2 +/+ mice.

Figure 9.

Effect of external Ca²⁺ on swelling-activated Cl⁻ and K⁺ currents in primary cultures of PCT cells from TASK2 +/+ mice. Under each experimental condition, the circled values represent the osmotic pressure in the pipette, and the values outside the circle represent the osmotic pressure in the extracellular bath solutions. (A) Characteristics of swelling-activated Cl⁻ currents activated by a hypotonic shock in the absence of external Ca²⁺. Whole-cell currents were recorded in solutions containing 140 mM NMDG-Cl both in the pipette and in the extracellular bath. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. (B) Characteristics of swelling-activated K⁺ currents activated by a hypotonic shock in the absence of external Ca²⁺. Whole-cell currents were recorded in solutions containing 125 mM of K⁺ in the pipette and 5 mM of K⁺ in the extracellular bath. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and +120 mV in 20-mV increments. (C) Histogram of current values at 100 mV of swelling-activated K⁺ currents in different conditions. Values were measured 200 ms after the onset of pulse. Each value is mean ± SEM of 10 cells obtained from five monolayers.

Figure 10.

Effect of hypotonicity in cultured DCT cells from TASK2 +/+ and TASK2 −/− mice. (A and B) Characteristics of swelling-activated whole-cell K⁺ currents in primary cultures of DCT cells from TASK2 +/+ and TASK2 −/− mice. Under each experimental condition, the circled values represent the osmotic pressure in the pipette, and the values outside the circle represent the osmotic pressure in the extracellular bath solutions. Whole-cell currents K⁺ were recorded in solutions containing 125 mM of K⁺ in the pipette and 5 mM of K⁺ in the extracellular bath. Membrane voltage was held at −50 mV and stepped to test potential values between −100 and 120 mV in 20-mV increments. (a) Whole-cell currents recorded in control conditions (350 mosmol/kg H₂O in the extracellular bath) after the mechanical rupture of membrane. (b) Whole-cell currents recorded 4–5 min after extracellular perfusion of a 290-mosmol/kg H₂O hypotonic solution. (c) Whole-cell currents were recorded 4 min after extracellular perfusion of a 350 mosmol/kg H₂O hypertonic solution. Values are means ± SEM of four different monolayers from four different mice. (C) RVD in cultured DCT cells from TASK2 +/+ and TASK2 −/− mice. After loading with Fura2, cultures were rinsed in 300 mosmol/kg H₂O NaCl solution, and fluorescence of Fura2 was measured at 360 nm for a control period of 1 min. Then, a hypotonic shock was induced by perfusing a 200-mosmol/kg H₂O NaCl solution. Images were recorded every 5 s. After analysis, relative volume change in percentage of initial volume was plotted against time as explained in materials and methods. Measurements were performed on six monolayers (25 random cells each) from TASK2 −/− and five monolayers (25 random cell each) from TASK2 +/+.