Volume 139, Issue 2, Pages 620-631 (August 2010) Dynamic Regulation of CFTR Bicarbonate Permeability by [Cl−]i and Its Role in Pancreatic Bicarbonate Secretion  Hyun Woo Park, Joo Hyun Nam, Joo Young Kim, Wan Namkung, Jae Seok Yoon, Jung–Soo Lee, Kyung Sik Kim, Viktoria Venglovecz, Michael A. Gray, Kyung Hwan Kim, Min Goo Lee  Gastroenterology  Volume 139, Issue 2, Pages 620-631 (August 2010) DOI: 10.1053/j.gastro.2010.04.004 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Measurements of [Cl−]i in the monolayers of human pancreatic duct cells. (A) The PANC1 human pancreatic duct cells expressing WT-CFTR and (B) CFPAC1 cells bearing the ΔF508 mutant CFTR were cultured on permeable supports to form monolayers. The double perfusion chamber with separate luminal (L) and basolateral (BL) perfusions was used to measure [Cl−]i in N-[ethoxycarbonylmethyl]-6-methoxy-quinolinium bromide–loaded cells. Activation of CFTR by cAMP stimulation (5 μmol/L forskolin and 100 μmol/L IBMX) augmented the luminal Cl− permeability in (A) PANC1 cells but not in (B) CFPAC1 cells. Gastroenterology 2010 139, 620-631DOI: (10.1053/j.gastro.2010.04.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Expression of [Cl−]i-sensitive kinases in human pancreas and their association with CFTR. Expression of (A) WNK1, (B) OSR1, and (C) SPAK in human pancreatic tissues was analyzed by immunoblotting and immunohistochemistry. Protein samples from HEK 293T cells transfected with plasmids expressing Myc-WNK1, Flag-OSR1, or Flag-SPAK were used as positive (+) controls and those from mock-transfected cells were used as negative (−) controls for immunoblotting. In immunohistochemistry, CFTR-positive (green) pancreatic duct cells highly coexpressed WNK1, OSR1, and SPAK (red). Gastroenterology 2010 139, 620-631DOI: (10.1053/j.gastro.2010.04.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Activation of OSR1/SPAK by Cl−-free solutions and the role of WNK1. The activation of OSR1 and SPAK was analyzed using antibodies against the phospho-T-loop. In A and B, HEK 293T cells transfected with plasmids expressing Myc-WNK1, Flag-OSR1, and Flag-SPAK were incubated in solutions containing various concentrations of Cl− and HCO3− for 40 minutes, and protein samples were collected. In some experiments, cells were incubated with hypotonic solution (*) (165 mOsm by reducing Na-gluconate concentrations) or bicarbonate-free, HEPES-buffered solution (**). In C and D, the role of WNK1 in the [Cl−]i-sensitive phosphorylation of OSR1 and SPAK was analyzed. HEK 293T cells transfected with the plasmids expressing Flag-OSR1 or Flag-SPAK were incubated with high HCO−/low Cl-containing solution for the indicated time. A batch of cells was transfected with the (C) Myc-WNK1 plasmid or (D) hWNK1 small interfering RNAs. In D (the lowermost lanes), a 5-fold larger amount of protein samples (100 μg) was loaded and a 2-fold longer immunoblot developing time was used to detect WNK1 endogenously expressed in HEK 293T cells. Gastroenterology 2010 139, 620-631DOI: (10.1053/j.gastro.2010.04.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 CFTR facilitates the low Cl−-containing solution-induced phosphorylation of OSR1 and SPAK. In A and B, HEK 293T cells were incubated in low Cl− -containing (0 mmol/L) solution for 40 minutes. Cotransfection with the plasmid expressing CFTR and cAMP stimulation (5 μmol/L forskolin and 100 μmol/L IBMX) induces the phosphorylation of OSR1 and SPAK in shorter time points. In C, HEK 293T cells expressing CFTR and indicated kinases were preincubated in the low Cl− -containing (0 mmol/L) solution for 40 minutes with cAMP stimulation, and then the solution was replaced to a standard 129-mmol/L Cl−-containing solution for the indicated time. In D, PANC1 cells expressing WT-CFTR and CFPAC1 cells bearing the ΔF508 mutant CFTR were incubated in low Cl−-containing (9 mmol/L) solution for up to 40 minutes with cAMP stimulation. Low Cl−-containing solution induces phosphorylation of OSR1 and SPAK in PANC1 cells but not in CFPAC1 cells. Gastroenterology 2010 139, 620-631DOI: (10.1053/j.gastro.2010.04.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Effects of WNK1 and SPAK activation on CFTR HCO3− permeability. Whole cell recordings were performed to measure CFTR HCO3− permeability by replacing the bath solution with high HCO3-containing (130 mmol/L) solution. Cells were stimulated with cAMP (5 μmol/L forskolin and 100 μmol/L IBMX) after establishing whole cell configuration. The current to voltage relationship (I–V curve) was obtained by depolarizing ramp pulses from −100 to 100 mV. In A and B, Erev was measured in HEK 293T cells (A) transfected with plasmids expressing CFTR only or (B) cotransfected with plasmids expressing CFTR, WNK1, and SPAK using pipettes containing 2 different concentrations of Cl− (140 mmol/L and 10 mmol/L). PHCO3/PCl was calculated according to the Goldman–Hodgkin–Katz equation as detailed in Materials and Methods. In C and D, the resting membrane potential (RMP) was measured in HEK 293T cells (C) transfected with plasmids expressing CFTR only or (D) cotransfected with plasmids expressing CFTR, WNK1, and SPAK using pipettes containing 10 mmol/L Cl−. Gastroenterology 2010 139, 620-631DOI: (10.1053/j.gastro.2010.04.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Low [Cl−]i increases CFTR HCO3− permeability in guinea pig pancreatic duct cells. Single pancreatic duct cells were freshly isolated from small intralobular and interlobular ducts and subjected to whole cell recordings. During resting membrane potential (RMP) measurement with zero-current clamp mode, I–V curve was obtained by applying ramp pulses from −100 to 100 mV after switching to the voltage clamp mode. Representative traces of RMP measurements using 150 mmol/L and 4 mmol/L Cl-containing pipette solutions are shown in A and C, respectively, and resulting I–V curves are shown in B and D. In A and C, ramp pulse signals generated by voltage clamping for I–V relationships were removed. From the basal current in guinea pig pancreatic duct cells (1), cAMP stimulation evoked an anion current that mainly exhibits a linear I-V relationship (2), is permeable to HCO3− (3), and is inhibited by CFTRinh-172 (4). Summarized results of PHCO3/PCl are presented in E. Gastroenterology 2010 139, 620-631DOI: (10.1053/j.gastro.2010.04.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Inhibition of CFTR-dependent, SLC26A3-mediated, and Slc26a6-mediated Cl−/HCO3− exchange activities by OSR1 and SPAK kinases. HEK 293T cells transfected with indicated plasmids were preincubated in Cl−-free solution for 40 minutes to activate OSR1 and SPAK kinases. All solutions contained 25 mmol/L HCO3− and were continuously gassed with 5% CO2. (A and B) CFTR-dependent Cl−/HCO3− exchange activities. Traces from cells transfected with indicated plasmids are presented in each panel. Summarized results are illustrated in B. Cells were stimulated with forskolin (Fosk, 5 μmol/L) and IBMX (100 μmol/L) to activate CFTR. Exposure to high Cl−-containing solution (129 mmol/L) in CFTR-activated cells evoked a rapid decrease in pHi due to Cl−o/HCO3−i exchange. (C and D) SLC26A3-mediated Cl−/HCO3− exchange activities. Traces from cells transfected with indicated plasmids are presented in each panel. Summarized results are illustrated in D. Exposure to high Cl−-containing solution in SLC26A3-transfected cells evoked Cl−o/HCO3−i exchange activity, which was insensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 300 μmol/L). (E and F) Slc26a6-mediated Cl−/HCO3− exchange activities. Mouse mSlc26a6 clone was used because it showed stronger Cl−/HCO3− exchange activity compared with human SLC26A6. Traces from cells transfected with indicated plasmids are presented in each panel. Summarized results are illustrated in F. Slc26a6-mediated Cl−/HCO3− exchange activities are inhibited by DIDS (300 μmol/L). Gastroenterology 2010 139, 620-631DOI: (10.1053/j.gastro.2010.04.004) Copyright © 2010 AGA Institute Terms and Conditions

Figure 8 A model for pancreatic HCO3− secretion. cAMP signals initially activate the CFTR-dependent Cl−/HCO3− exchange at the apical membrane of pancreatic duct cells. A gradual increase in luminal HCO3− level and decline in Cl− level induces a decrease in [Cl−]i due to the apical CFTR Cl− channel activity. This in turn activates WNK1 and the downstream OSR1 and SPAK kinases. Activation of OSR1 and SPAK increases CFTR HCO3− permeability, making CFTR primarily an HCO3− channel, and inhibits the activity of apical Cl−/HCO3− exchange. These combinatorial functions maintain greater than 140 mmol/L HCO3− concentration in the human pancreatic juice (see text for details). Gastroenterology 2010 139, 620-631DOI: (10.1053/j.gastro.2010.04.004) Copyright © 2010 AGA Institute Terms and Conditions