Volume 118, Issue 6, Pages 1187-1196 (June 2000) Cystic fibrosis transmembrane conductance regulator currents in guinea pig pancreatic duct cells: Inhibition by bicarbonate ions  Catherine M. O'Reilly, John P. Winpenny, Barry E. Argent, Michael A. Gray  Gastroenterology  Volume 118, Issue 6, Pages 1187-1196 (June 2000) DOI: 10.1016/S0016-5085(00)70372-6 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Biophysical characteristics of whole-cell Cl− currents recorded from guinea pig pancreatic duct cells. (A-C) CFTR. (D-F) Cltv-dep. (A and D) Basal currents. (B and E) currents recorded from the same cells after exposure to the cAMP cocktail. Note the different vertical scales for CFTR and Cltv-dep currents. (B) CFTR currents have time- and voltage-independent kinetics. (E) In contrast, Cltv-dep currents have time- and voltage-dependent kinetics and exhibit clear tail currents. (C and F) I/V plots for the current traces shown. Note that the CFTR plot (C) is near linear and the Cltv-dep plot (F) is outwardly rectified. Gastroenterology 2000 118, 1187-1196DOI: (10.1016/S0016-5085(00)70372-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Dose-response relationships for the effect of secretin and forskolin on whole-cell Cl− current density. (A-C) Data for CFTR cells; (D-F) data for Cltv-dep cells. (A) Cumulative dose-response curves for the effect of secretin (closed symbols) and forskolin (open symbols) on CFTR cells. These cells exhibited only CFTR currents. The inward CFTR current densities recorded at −60 mV are plotted, after subtraction of basal currents, for individual cells (4 cells with secretin and 3 cells with forskolin) as a percentage of the response to either 10−8 mol/L secretin or 10−5 mol/L forskolin. Basal current densities were not significantly different (secretin group, 5.1 ± 1.3 pA/pF [n = 4 cells]; forskolin group, 2.4 ± 1.0 pA/pF [n = 3 cells]). (B1 and B2) Currents from the secretin-stimulated cell indicated by the closed triangles and arrow in A obtained with 10−9 mol/L (B1) and 10−8 mol/L secretin (B2). (C1 and C2) Currents from the forskolin-stimulated cell indicated by the open triangles and arrow in A obtained with 10−6 mol/L (B1) and 10−5 mol/L forskolin (B2). (D) Cumulative dose-response curves for the effect of secretin (closed symbols) and forskolin (open symbols) on Cltv-dep cells. The inward Cltv-dep current densities recorded at −60 mV are plotted for individual cells (4 cells with secretin and 3 cells with forskolin). Basal currents were not significantly different for secretin group (6.2 ± 1.3 pA/pF; n = 4 cells) and forskolin group (5.9 ± 3.7 pA/pF; n = 3 cells). Most of these cells (3 of 4 with secretin and 2 of 3 with forskolin) exhibited CFTR currents at low doses of the stimulants and Cltv-dep currents at higher doses. (E and F) Currents recorded from cells that converted between the 2 current types. (E1 and E2) Currents from the secretin-stimulated cell indicated by the closed circles and arrow in D obtained with 10−10 mol/L (E1) and 10−9 mol/L secretin (E2). (F1 and F2) Currents from the forskolin-stimulated cell indicated by the open squares and arrow in D obtained with 5 × 10−7 mol/L (F1) and (E2, F2) 10−5 mol/L forskolin (F2). E1 and F1 are CFTR currents, whereas E2 and F2 are Cltv-dep currents. Note the difference in magnitudes of the Cltv-dep and the CFTR currents (B1, B2, C1, C2, E1, F1). When performing the cumulative dose-response curves (A and D), the total time of exposure to either secretin or forskolin was about 30 minutes for all cells. To obtain the current traces in sections B, C, E, and F, cells were voltage clamped over the range −100 mV to 100 mV in 20-mV steps. Gastroenterology 2000 118, 1187-1196DOI: (10.1016/S0016-5085(00)70372-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Inhibitory effect of HCO3− on CFTR and Cltv-dep currents. The duct cells were stimulated with cAMP cocktail throughout the experiments, and the currents were sampled at Vm of ±60 mV. Upward deflections of the traces are outward currents (Cl− influx) flowing at +60 mV, and downward deflections are inward currents (Cl− efflux) flowing at −60 mV. The normal bath solution was switched to one containing nominally 100 mmol/L HCO3− (replacement of 100 mmol/L Cl− in the normal bath solution) at the arrow marked HCO3−. The normal bath solution was restored at the arrow marked wash. The pulse protocol was interrupted at various points to collect I/V data. Note that the fast onset of HCO3− inhibition and its slow reversal may reflect the anion's relatively low Ki value ( ̃7 mmol/L) and the kinetics of solution exchange in the tissue bath. To establish a half-inhibitory concentration of HCO3− in the bath ( ̃7 mmol/L) would require only 7% exchange of the bath Cl− solution with the 100 mmol/L HCO3− solution. In contrast, for half-recovery, 93% of the 100 mmol/L HCO3− in the bath would have to be exchanged for Cl−. Gastroenterology 2000 118, 1187-1196DOI: (10.1016/S0016-5085(00)70372-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Dose-response curves for the inhibitory effect of external HCO3− on CFTR and Cltv-dep currents. ■, CFTR; ●, Cltv-dep. The actual HCO3− concentration in the bath solutions was measured using a blood gas analyzer. Inward currents were measured at Erev −60 mV so that the electrochemical driving force on the HCO3− remained constant. Data points (mean ± SE) contain observations from between 8 and 14 cells. The curves have been fit to a Michaelis–Menten equation: I = Imaximum × [HCO3−]/Ki + [HCO3−], where I is percentage inhibition, Imaximum is the maximum percentage inhibition, and Ki is the bicarbonate concentration giving half-maximal inhibition. Gastroenterology 2000 118, 1187-1196DOI: (10.1016/S0016-5085(00)70372-6) Copyright © 2000 American Gastroenterological Association Terms and Conditions