Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells

Abstract

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3, 7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl(-) current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.

The Cystic Fibrosis Mutation G551D Alters the Non-Michaelis-Menten Behavior of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel and Abolishes the Inhibitory Genistein Binding Site

“…The currents reversed at Ϫ40 mV, as shown in Fig. 6D, a value close to the theoretical Cl Ϫ equilibrium potential imposed by our conditions (E Ce Ϫ ϭ Ϫ42 mV, see "Experimental Procedures"), confirming the chloride nature of the current (11,12). The genistein-activated conductance had a current density of 16.42 Ϯ 3.77 pA/picofarads (n ϭ 15) and was statistically different from control conditions (1.92 Ϯ 0.23, pA/picofarads, n ϭ 17, p Ͻ 0.001) when measured at ϩ40 mV (Fig.…”

“…Iodide Efflux Experiments-CFTR chloride channel activity was assayed by measuring the rate of iodide ( 125 I) efflux versus time from cells as described previously (11,12). All experiments were performed at 37°C.…”

Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

“…The gating defect of ⌬F508, G551D, and G1349D can be corrected pharmacologically by small molecules called CFTR potentiators (Galietta and Moran, 2004;Verkman et al, 2006). Such compounds, which include flavonoids (genistein, apigenin), fluorescein derivatives, xanthines, benzimidazolones, phenylglycines, sulfonamides, 1,4-dihydropyridines, and tetrahydrobenzothiophenes (Illek et al, 1999;Bulteau et al, 2000;Al-Nakkash et al, 2001;Cai and Sheppard, 2002;Yang et al, 2003;Pedemonte et al, 2005a,b), enhance mutant CFTR activity through a decrease in the time spent by the channel in the closed state (Pedemonte et al, 2005a,b). The mechanism of action of CFTR potentiators is unknown but it has been hypothesized that they bind CFTR at the level of NBDs, possibly at the interface between them (Zegarra- Moran et al, 2007), thus promoting NBD dimerization and favoring CFTR opening.…”

Discovery of α-Aminoazaheterocycle-Methylglyoxal Adducts as a New Class of High-Affinity Inhibitors of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

“…Among the known inhibitors of Cl Ϫ channels are stilbene acids such as 4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonic acid and 4-acetamido-4Ј-isothiocyanostilbene-2,2Ј-disulfonic acid, carboxylic acids such as diphenylamine-2-carboxylic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, sulfonylureas glibenclamide and tolbutamide, and indanyloxyacetic acid IAA-94 (Schultz et al, 1999). The calixarene derivative 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxycalix[4]arene developed after screening and chemical synthesis was the first nanomolar affinity inhibitor of the volume-regulated Cl Ϫ channels (Singh et al, 1995;Bulteau et al, 2000). More recently, two novel and more selective CFTR inhibitors with low micromolar affinity, thiazolidinone and glycine hydrazide agents, were discovered (Ma et al, 2002;Muanprasat et al, 2004).…”