Direct block of the cystic fibrosis transmembrane conductance regulator Cl⁻ channel by niflumic acid

Abstract

Niflumic acid is widely used to inhibit Ca²⁺-activated Cl⁻ channels. However, the chemical structure of niflumic acid resembles that of diphenylamine-2-carboxylate, a drug that inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel. To investigate how niflumic acid inhibits the CFTR Cl⁻ channel, we studied recombinant wild-type human CFTR in excised inside-out membrane patches. When added to the intracellular solution, niflumic acid caused a concentration- and voltage-dependent decrease of CFTR Cl⁻ current with half-maximal inhibitory concentration (K_i) of 253 μM and Hill co-efficient of ∼1, at −50 mV. Niflumic acid inhibition of single CFTR Cl⁻ channels was characterised by a very fast, flickery block that decreased dramatically current amplitude without altering open-probability. Consistent with these data, spectral analysis of CFTR Cl⁻ currents suggested that channel block by niflumic acid was described by the closed ↔ open ↔ blocked kinetic scheme with blocker on rate (k_on)=13.9×10⁶ M⁻¹ s⁻¹, off rate (k_off)=3348 s⁻¹ and dissociation constant (K_d)=241 μM, at −50 mV. Based on these data, we tested the effects of niflumic acid on transepithelial Cl⁻ secretion and cyst growth using type I MDCK epithelial cells. Niflumic acid (200 μM) inhibited cAMP-stimulated, bumetanide-sensitive short-circuit current by 55%. Moreover, the drug potently retarded cyst growth. We conclude that niflumic acid is an open-channel blocker of CFTR that inhibits Cl⁻ permeation by plugging the channel pore. It or related agents might be of value in the development of new therapies for autosomal dominant polycystic kidney disease.

• chloride ion channel
• channel block
• chloride secretion
• cyst growth
• non-steriodal anti-inflammatory drug

ADPKD, autosomal dominant polycystic kidney disease; A9C, anthracene-9-carboxylate; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid; DPC, diphenylamine-2-carboxylate; f_c, corner frequency; i, single-channel current amplitude; I_sc, short-circuit current; M, transmembrane segment; MDCK cells, Madin Darby canine kidney cells; NMDG, N-methyl-D-glucamine; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; NSAID, non-steroidal anti-inflammatory drug; P_o, open-probability; R_t, transepithelial resistance; TES, N-tris[Hydroxymethyl]methyl-2-aminoethanesulphonic acid

ADPKD, autosomal dominant polycystic kidney disease; A9C, anthracene-9-carboxylate; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid; DPC, diphenylamine-2-carboxylate; f_c, corner frequency; i, single-channel current amplitude; I_sc, short-circuit current; M, transmembrane segment; MDCK cells, Madin Darby canine kidney cells; NMDG, N-methyl-D-glucamine; NPPB, 5-nitro-2-(3-phenylpropylamino)-benzoic acid; NSAID, non-steroidal anti-inflammatory drug; P_o, open-probability; R_t, transepithelial resistance; TES, N-tris[Hydroxymethyl]methyl-2-aminoethanesulphonic acid