Fig 2: Inhibition of PLC, PI3-K, and phospholipase A2 signaling cascades does not affect regulation of ClC-K2 activity by Ang II.A, representative continuous current trace from a cell-attached patch monitoring activity of basolateral ClC-K2 chloride channels in an intercalated cell of a freshly isolated collecting duct in the control, upon treatment with PLC inhibitor, U73122 (10 µM, gray line), Angiotensin II (500 nM, black line) in the continued presence of the blocker, and following washout with the control medium. The patch was clamped to -Vp = -60 mV; “c” denotes closed nonconducting state. Areas (1, control) and (2, Ang II + U73122) are shown below at an expanded timescale. Summary graphs of changes in ClC-K2 open probability (Po) upon pretreatment with PLC blocker, 10 µM U73122 (B), PI3-K blocker, 20 µM LY294002 (C), phospholipase A2 blocker, 30 µM AACOCF3 (D); Ang II application in the continued presence of the respective antagonist, and following washout with control medium, as similarly shown in paired patch clamp experiment in (A). * - significant increase (p < 0.05) versus pretreatment with respective blocker (one-way ANOVA); ‡ - significant decrease (p < 0.05) versus control (one-way ANOVA). Collecting ducts from at least three different mice were used for each set of experiments. PLC, phospholipase C.

Fig 3: Ang II stimulates activity of ClC-K2 channel in a dose-dependent manner.A, representative continuous current trace from a cell-attached patch monitoring activity of basolateral 10 pS ClC-K2 chloride channels in an intercalated cell in a freshly isolated collecting duct in the control, upon application of 500 nM Ang II (shown with a line on top) and following washout with control medium. The patch was clamped to -Vp = -60 mV; “c” denotes closed nonconducting state. Areas (1, control) and (2, Ang II) are shown below at an expanded timescale. B, summary graph of changes in ClC-K2 open probability (Po) upon treatment with 500 nM Ang II from paired patch clamp experiments similar to that shown in (A). C, summary graph of average changes in ClC-K2 Po in individual cells upon application of different Ang II concentrations. * - significant increase (p < 0.05) versus control (one-way ANOVA). Collecting ducts from at least three different mice were used for each set of experiments.

Fig 4: Ang II increases ClC-K2 activity in intercalated cells by acting on AT1receptors.A, representative continuous current trace from a cell-attached patch monitoring activity of basolateral ClC-K2 chloride channels in an intercalated cell of a freshly isolated collecting duct in the control, upon treatment with AT1 receptor blocker losartan (1 µM, gray line), and Ang II (500 nM, black line) in the continued presence of the antagonist. The patch was clamped to -Vp = -60 mV; “c” denotes closed nonconducting state. Areas (1, control) and (2, Ang II + losartan) are shown below at an expanded timescale. B, summary graph of changes in ClC-K2 open probability (Po) upon treatment with losartan and following Ang II in paired patch clamp experiments similar to that shown in (A). C, summary graph of changes in ClC-K2 Po in the control, during treatment with AT2 receptor agonist, CGP42112 (100 nM for 3 min), and following washout with control medium. Collecting ducts from at least three different mice were used for each set of experiments.

Fig 5: Deletion of AT1receptors decreases renal ClC-K2 expression.A, representative Western blot from whole kidney lysates of WT and AT1R -/- mice probed with anti-ClC-K antibodies. The Ponceau red staining of the same nitrocellulose membrane demonstrating equal protein loading is shown in the bottom panel. B, summary graph comparing ClC-K expression levels in WT and AT1R -/- mice. The intensity values were normalized to the total signal of the respective lines in Ponceau red staining. The number of individual mice for each experimental condition is shown. Both SEM (smaller bars) and SD (larger bars) are shown. * - significant decrease (p < 0.05) versus control (one-way ANOVA).