The arylaminobenzoate niflumic acid (NFA) is widely used to inhibit Ca2+-activated Cl– channels (White & Aylwin, 1990). NFA shares a high degree of structural homology with another arylaminobenzoate, diphenylamine-2-carboxylate (DPC), which inhibits a variety of Cl– channels including the cystic fibrosis transmembrane conductance regulator (CFTR; McCarty et al. 1993).
We investigated the effect of NFA on the CFTR Cl– channel using inside-out membrane patches excised from C127 cells stably expressing wild-type human CFTR (Lansdell et al. 2000). The pipette (external) solution contained 10 mM Cl– and the bath (internal) solution contained 147 mM Cl–, 0.3-5 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 performed statistical analyses using Student’s paired t tests. Addition of NFA to the intracellular solution caused a concentration-dependent decrease of CFTR Cl– current with half-maximal inhibitory concentration (Ki) of 253 ± 16 mM and Hill coefficient of 0.95 ± 0.03 (n = 5); inhibition was readily reversible.
At the single-channel level, NFA (200 mM) caused a very fast, flickery block of CFTR that dramatically decreased current amplitude (i; control, -0.80 ± 0.03 pA; NFA (200 mM), -0.36 ± 0.04 pA; n = 5; P < 0.01), but was without effect on open probability (n = 5; P > 0.35). To investigate how NFA inhibits CFTR, we performed experiments to distinguish between allosteric and open-channel block. CFTR inhibition by the allosteric blocker genistein is relieved by elevated concentrations of ATP (Lansdell et al. 2000). In the presence of ATP (0.3 and 5 mM), NFA (200 mM) decreased CFTR Cl– current to 43 ± 2 and 49 ± 2 % of the control value (n = 6), respectively. These data suggest that NFA does not inhibit CFTR by an allosteric mechanism. To learn whether NFA is an open-channel blocker of CFTR, we examined the voltage dependence of NFA inhibition of CFTR. When the external [Cl–] was 10 mM, the voltage-dependent dissociation constant (Kd) for NFA inhibition of CFTR decreased from 391 ± 60 mM at 0 mV to 179 ± 23 mM at -100 mV (P < 0.05, n = 5) and the electrical distance sensed by NFA (δ) was 0.20 ± 0.03 (n = 5) when measured from the intracellular side of the membrane over the voltage range -100 to -40 mV. These data indicate that NFA inhibition of CFTR is voltage dependent and that the drug binding site is located within the electric field of the membrane. These data suggest that NFA is an open channel blocker of CFTR.
This work was supported by the CF Trust and NKRF.