Lumen. -60 mv, ph 7.3 Cl - (E Cl CFTR HCO mv, ph 7.3 HCO HCO. Channel or Other electrogenic HCO 3- up to a 80-mM concentration when [Cl - ] i
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1 . Proximal Duct 1 Cl HCO 8 Cl 8 HCO Lumen CFTR AE 6 mv, ph 7. Cl (E Cl = 712 ) Cl 2 HCO 2 lood ph HCO. Distal Duct 2 Cl 128 HCO >14 HCO? 6 mv, ph 7. Cl (E Cl = 47 ) Cl HCO HCO 5 2 HCO HCO Channel or Other electrogenic HCO transporter? Supplementary Figure 1. A schematic diagram of HCO secretion that can be maximally achieved by the Cl /HCO exchange at the apical membrane of pancreatic duct cells. (A) The 1:1 Cl /HCO exchange can secrete HCO up to a 8 concentration when [Cl ] i and [HCO ] i are around same levels. () When [Cl ] i decreases to a 5 level, the apical Cl /HCO exchanger can secrete HCO up to a 128 concentration. A HCO channel or other electrogenic mechanisms would be one of the strong candidates for the transporter responsible for high HCO secretion at the apical membrane of pancreatic duct cells. A HCO channel can secrete HCO up to a 2 concentration when cells maintain a 6 mv membrane potential according to the Nernst equation.
2 [Cl ] L PANC1 (WTCFTR) [HCO ] L μm Forskolin 1 μm IMX 1 μm CFTRinh172 [Cl ] i min [Cl ] L [HCO ] L [Cl ] L CFPAC1 (ΔF58CFTR) [HCO ] L μm Forskolin 1 μm IMX 1 μm CFTRinh172 4 [Cl ] i min [Cl ] L [HCO ] L Supplementary Figure 2. Inhibition of luminal Cl permeability by the CFTR Cl channel inhibitor CFTRinh172. Measurements of [Cl ] i in the monolayers of PANC1 cells expressing WTCFTR (A) and CFPAC1 cells bearing F58 mutant CFTR () were performed using similar protocols described in the legend for Figure 1. Inhibition of the luminal Cl permeability by CFTRinh172 (1 µm) indicates that CFTR Cl channel mediates most of the Cl permeability in the luminal membrane of PANC1 cells. L: luminal side, L: basolateral side, IMX: isobutyl1methylxanthine.
3 CFTR MycWNK1 MycWNK4 Resting Forskolin IMX Resting Forskolin IMX CFTR FlagOSR1 FlagSPAK Immunoprecipitate IP: AntiMyc, lot: AntiCFTR IP: AntiFlag, lot: AntiCFTR lot: AntiCFTR lot: AntiCFTR Cell Lysate lot: AntiMyc (WNK1) lot: AntiFlag SPAK OSR1 lot: AntiMyc (WNK4) Supplementary Figure. Coimmunoprecipitation (IP) was performed in HEK T cells transiently transfected with plasmids expressing CFTR, MycWNK1 (A), MycWNK4 (A), FlagOSR1 (), and/or FlagSPAK (). In each righthand panel of A and, IPs were performed in cells incubated with 5 µm forskolin and 1 µm IMX. For immunoblotting, 2 µg of protein was loaded into each lane, and IP was performed with a total of 1 mg lysate.
4 Cl 15 CFTR only Fork 5 µm IMX 1 µm HCO 146 Cl time (sec) Pipette Cl 1 : P HCO /P Cl =.27 CFTRinh172 1 µm voltage (mv) C Cl 15 CFTR WNK1 SPAK Fork 5 µm IMX 1 µm HCO 146 Cl time (sec) CFTRinh172 1 µm Pipette Cl 1 : P HCO /P Cl = 1.2 D voltage (mv) Supplementary Figure 4. Inhibition of CFTR HCO permeability by the CFTR channel inhibitor CFTRinh172. Resting membrane potential (RMP) and IV curves were obtained in HEK T cells transfected with plasmids expressing CFTR only (A and ), or cotransfected with plasmids expressing CFTR, WNK1, and SPAK (C and D) using protocols described in the legend for Figure 5. IV curves were obtained by applying ramp pulses during the RMP measurements. In each panel, camp stimulation (forskolin IMX) evoked an anion current that has a linear IV relationship (), is permeable to HCO (), and is inhibited by CFTRinh172 (). CFTRinh172 (1 µm) inhibited the HCO mediated CFTR current by an average of 72.1 ± 2.7 % (n=16) at 7 mv.
5 Main Pancreatic Duct Duct Cells Used for Patch Clamp rd ranch 1 st ranch 2 nd ranch X 4 Supplementary Figure 5. Isolation of single pancreatic duct cells for whole cell current measurement. (A) Guinea pig pancreatic tissues were partially digested with collagenase (type II,.5 mg/ml; Serva, Heidelberg, Germany). Small interlobular or intralobular ducts proximal to the acini ( third branch from the main duct) were then isolated by microdissection using stainless steel needles under a dissecting microscope at x 4 magnification. () The isolated ducts were further digested with elastase (5 U/ml, Sigma, St.Louis, MO) and teased apart using stainless steel needles in order to dissociate into single cells. Single pancreatic duct cells were subjected to whole cell current measurement.
6 PANC1 PANC1 PANC1 siwnk1 Scr Scr P <.1 P <.1 15 KDa AntiWNK AntiSPAK P HCO / P Cl AntiCFTR 5 AntiOSR1 n=6 n=1 n=4 Pipette: Cl siwnk1 Scr Scr Supplementary Figure 6. Low [Cl ] i increases CFTR HCO permeability in PANC1 cells. P HCO /P Cl was measured in PANC1 cells using the protocols described in the legend for Figure 5. (A) PANC1 cells that endogenously express WTCFTR, WNK1, SPAK, and OSR1 were treated with sirnas directed against WNK1. The WNK1 sirna, a mixture of four sirnas against hwnk1, and control scrambled sirna (Scr) were purchased from Dharmacon. sirnas were transiently transfected into cells using Lipofectamine 2 reagents (Invitrogen) and immunoblotting was performed 2 days after sirna transfection. () Wholecell recordings were performed to measure CFTR HCO permeability. Decreasing pipette Cl from 14 to 4 increased P HCO /P Cl from.69 ±. to 2.42 ±.. Importantly, this low [Cl ] i induced increase in P HCO /P Cl was abolished by the transfection with sirna directed against WNK1, indicating that WNK1 is essential in this process. The CFTR anion channel in PANC1 cells that is activated by camp (forskolin 5 µm IMX 1 µm) and inhibited by CFTRinh172 (1 µm) has a higher basal P HCO /P Cl value (.69) than those of CFTRtransfected HEK T cells (.9) and guinea pig pancreatic duct cells (.24). Moreover, P HCO /P Cl in PANC1 cells can reach up to 2.42 in the 4 Cl containing pipette. The reasons for this high P HCO /P Cl are unknown. The relatively small expression of CFTR and high expression of WNK1 in PANC1 cells, thus a high WNK1/CFTR expression ratio, could be one of the reasons.
7 CFTR () SLC26A () WNK1 () [Cl ] o ph i C 2 min Fosk IMX CFTR () Slc26a6 () WNK1 () 2 min Fosk IMX CFTR () SLC26A () WNK1 () Fosk IMX CFTR () Slc26a6 () WNK1 () Fosk IMX CFTR () SLC26A () WNK1 () OSR1 () Fosk IMX CFTR () Slc26a6 () WNK1 () OSR1 () Fosk IMX CFTR () SLC26A () WNK1 () SPAK () Fosk IMX CFTR () Slc26a6 () WNK1 () SPAK () Fosk IMX CFTR SLC26A WNK1 OSR1 SPAK D n=4 n= n=4. n= n= n=4 CFTR Slc26a6 WNK1 OSR1 SPAK Supplementary Figure 7. Inhibition of SLC26A and Slc26a6mediated Cl /HCO exchange activities by OSR1 and SPAK kinases in CFTR cotransfected cells. Cl /HCO exchange activities were measured in HEK T cells transfected with indicated plasmids using protocols described in the legend for Figure 7. Representative traces from cells expressing SLC26A with CFTR and Slc26a6 with CFTR are presented in panels A and C, respectively. A summary of multiples experiments is shown in panels and D. Similar to the results in Figures 7C7F, total Cl /HCO exchange activities from cells expressing SLC26A or Slc26a6 were also inhibited by the activation of OSR1 and SPAK in CFTR cotransfected cells.
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