Cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in the secretion of a HCO3–-rich fluid by the ductal tree of the pancreas. We have previously shown, using fast whole cell recording (WCR), that Cl– efflux through CFTR is inhibited when extracellular Cl– is replaced with various anions, including HCO3– (O’Reilly et al. 2000). The degree of inhibition of Cl– efflux is not voltage dependent, nor is it related to the relative permeability of the replacement anion, suggesting that an intrapore effect is unlikely. The aim of this study was to investigate the mechanism by which Cl– efflux through CFTR is inhibited. The experiments were performed on CHO cells stably transfected with human CFTR.
Iso-osmotic replacement of external NaCl (155.5 mM down to 6.5 mM) with mannitol in fast WCR caused a dose-dependent inhibition of inward current, in a manner identical to the effects observed following anion replacement. This indicates that a reduction in external [Cl–] alone is sufficient to produce the inhibitory effect. Comparable results were obtained using the slow WCR configuration, but the inhibition caused by reducing external [Cl–] was significantly less under these conditions over a concentration range (Student’s unpaired t-test, P < 0.05). For example, when extracellular Cl– was lowered from 155.5 mM to 116.5, 91.5 or 71.5 mM (mannitol replacement) the mean percentage inhibitions during fast WCR were 36.3 ± 3.6, 43.6 ± 8.7 and 59.1 ± 7.6, respectively (mean ± S.E.M., n = 4-6), compared to 12.2 ± 4.4, 7.2 ± 1.6 and 24.4 ± 2.3, respectively, during slow WCR. Overall, the dose-response curve for slow WCR is shifted to the left by ~50 mM, suggesting that loss of a cytosolic factor (in fast WCR) increases the inhibition. A reduction in internal [Cl–], from 114 down to 54 mM, did not affect the peak stimulated Cl– current density, nor alter the subsequent inhibition of efflux by extracellular Cl– replacement. The addition of GTP (0.2 mM) to the pipette solution during fast WCR also had no effect on the inhibition of Cl– efflux. However, increasing the internal [Ca2+] from the control value of 1 nM to 100 nM during fast WCR reduced the degree of inhibition to levels comparable with slow WCR. Under these conditions, the mean percentage inhibition of the inward current was 9.3 ± 8, 23.5 ± 3.5 and 30.7 ± 5 at 116.5, 91.5 and 71.5 mM external [Cl–], respectively.
These data suggest that intracellular [Ca2+] can modulate the inhibitory effect of extracellular Cl– replacement on Cl– efflux through CFTR. In the pancreatic duct, changes in luminal [Cl–] may act as a signal that couples changes in the luminal environment to the secretory activity of CFTR.
This work was supported by the Wellcome Trust.