The loop diuretic furosemide is widely used to inhibit the Na⁺-K⁺-2Cl^– cotransporter (Haas & Forbush, 2000). However, Venglarik (1997) demonstrated that furosemide inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel. To investigate the mechanism of furosemide inhibition of the CFTR Cl^– channel, we used inside-out membrane patches excised from C127 cells stably expressing wild-type human CFTR (for Methods, see Lansdell et al. 2000). The pipette (external) solution contained 10 mM Cl^– and the bath (internal) solution contained 147 mM Cl^–, 0.3 or 1 mM ATP and 75 nM PKA at 37 °C; voltage was -50 mV. We expressed data as means ± S.E.M. of n observations and we performed statistical analyses using Student’s unpaired t test.

When added to the internal solution, furosemide (100 µM) caused a flickery block of single CFTR Cl^– channels that decreased both open probability (P_o; control, 0.41 ± 0.03; furosemide (100 µM), 0.17 ± 0.05; n = 6), and single-channel current amplitude (i; control, -0.76 ± 0.01 pA; furosemide (100 µM), -0.63 ± 0.03 pA; n = 6). Block was readily reversible on washing (n = 6). To learn whether furosemide is an open-channel blocker of CFTR, we examined the voltage dependence of furosemide inhibition. When excised patches were bathed in symmetrical 147 mM Cl^– solutions, furosemide (100 µM) strongly inhibited CFTR Cl^– currents at negative voltages. However, channel block was completely relieved at positive voltages. Using current values in the absence and presence of furosemide, the voltage-dependent dissociation constant (K_d) for furosemide inhibition at 0 mV was calculated to be 332 ± 29 µM (n = 5). This voltage dependence of inhibition suggests that furosemide binds within the electric field of the membrane possibly within the channel pore. If the binding site is located within the channel pore, the passage of Cl^– ions through the channel pore would be predicted to interfere with furosemide inhibition. Consistent with this idea, when the external [Cl^–] was reduced to 10 mM, the K_d of furosemide inhibition at 0 mV decreased to 156 ± 15 µM (n = 6; P < 0.01).

These results suggest that furosemide is an open-channel blocker of the CFTR Cl^– channel. They also suggest that furosemide and Cl^– ions might compete for a common binding site.

This work was supported by the CF Trust and NKRF.