The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl– channel with complex regulation. We previously demonstrated that intracellular pH has multiple effects on the activity of CFTR (Chen et al. 2002). To elucidate how pH modulates the activity of CFTR, we investigated the effects of voltage on the CFTR Cl– channel at different values of intracellular pH using excised inside-out membrane patches from C127 cells stably expressing wild-type human CFTR (for Methods, see Lansdell et al. 2000).
Membrane patches were bathed in symmetrical 147 mM Cl– solutions and the bath (internal) solution contained PKA (75 nM) and ATP (1 mM) at 37 °C. To adjust the bath solution to pH 8.3 and pH 6.3, we used Tris and H2SO4, respectively. Data are means ± S.E.M. of n observations and statistical analyses were performed using Student’s paired t test.
We began by studying the effect of voltage on the inhibition of CFTR Cl– currents at pH 8.3. At pH 8.3, CFTR Cl– currents were decreased by equivalent amounts at negative and positive voltages (e.g. -100 mV: pH 7.3, 100% pH 8.3, 75 ± 5% +100 mV: pH 7.3, 100% pH 8.3, 75 ± 4% n = 4). Consistent with these data, at pH 8.3 the open probability (Po) of CFTR was decreased at both negative voltages (e.g. -80 mV: pH 7.3, Po = 0.42 ± 0.004; pH 8.3, Po = 0.32 ± 0.01; n = 5; P < 0.01) and positive voltages (e.g. +80 mV: pH 7.3, Po = 0.42 ± 0.02; pH 8.3, Po = 0.34 ± 0.01; n = 5; P < 0.01). However, at pH 8.3 there was a small, but significant, decrease in single-channel current amplitude (i) at negative voltages (e.g. -80 mV: pH 7.3, -1.00 ± 0.02 pA; pH 8.3, -0.92 ± 0.03 pA; n = 5; P < 0.01). This decrease in i was relieved at positive voltages (e.g. +80 mV: pH 7.3, -0.95 ± 0.03 pA; pH 8.3, -0.95 ± 0.03 pA; n = 5; P = 0.48).
Next, we studied the effect of voltage on the stimulation of CFTR Cl– currents at pH 6.3. Voltage was without effect on i at pH 6.3 either at negative or positive voltages. Similarly, at pH 6.3 the increase of Po was also voltage independent (e.g. -80 mV: pH 7.3, 0.43 ± 0.01; pH 6.3, 0.65 ± 0.03; n = 6; P < 0.01 and +80 mV: pH 7.3, 0.44 ± 0.02; pH 6.3, 0.64 ± 0.01; n = 6; P < 0.01).
In conclusion, our data demonstrate that pH-dependent changes in Po are voltage independent, but that the decrease in i at pH 8.3 is voltage dependent. These data suggest that intracellular pH might modulate CFTR activity primarily by altering the function of the cytoplasmic domains of CFTR that control channel gating.
This work was supported by the CF Trust and the University of Bristol.