Renaud Dérand

Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

Activation of VPAC1 receptors by VIP and PACAP-27 in human bronchial epithelial cells induces CFTR-dependent chloride secretion

In the human airway epithelium, VIP/PACAP receptors are distributed in nerve fibers and in epithelial cells but their role in transepithelial ion transport have not been reported. Here, we show that human bronchial epithelial Calu‐3 cells expressed the VPAC1 receptor subtype which shares similar high affinity for VIP and PACAP‐27. The stoichiometric binding parameters characterizing the 125I‐VIP and 125I‐PACAP‐27 binding to these receptors were determined. We found that VIP (EC50∼7.6 nM) and PACAP‐27 (EC50∼10 nM) stimulated glibenclamide‐sensitive and DIDS‐insensitive iodide efflux in Calu‐3 cells. The protein kinase A (PKA) inhibitor, H‐89 and the protein kinase C (PKC) inhibitor, chelerythrine chloride prevented activation by both peptides demonstrating that PKA and PKC are part of the signaling pathway. This profile corresponds to the pharmacological signature of CFTR. In the cystic fibrosis airway epithelial IB3‐1 cell lacking functional CFTR but expressing VPAC1 receptors, neither VIP, PACAP‐27 nor forskolin stimulated chloride transport. Ussing chamber experiments demonstrated stimulation of CFTR‐dependent short‐circuit currents by VIP or PACAP‐27 applied to the basolateral but not to the apical side of Calu‐3 cells monolayers. This study shows the stimulation in human bronchial epithelial cells of CFTR‐dependent chloride secretion following activation by VIP and PACAP‐27 of basolateral VPAC1 receptors.

Comparative pharmacology of the activity of wild-type and G551D mutated CFTR chloride channel: effect of the benzimidazolone derivative NS004.

The pharmacological activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel mutated at glycine 551 (G551D-CFTR) was studied in the presence of the benzimidazolone derivative NS004 and compared to that of wild-type (wt) CFTR. Using iodide (125I) efflux and whole-cell patch-clamp techniques we found dose-dependent stimulation of phosphorylated wt-CFTR channels by NS004 with an EC50 ≈ 11 µM. With non-phosphorylated CFTR, the effect of NS004 was apparent only at concentration >100 µM. In G551D-CFTR-expressing CHO cells, neither forskolin (from 0.1 to 10 µM) nor NS004 (from 0.1 to 200 µM) added separately were able to stimulate channel activity. However, in the presence of 10 µM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC50 ≈ 1.5 µM. We also determined the half-maximal effective concentration of forskolin (EC50 ≈ 3.2 µM) required to stimulate G551D channel activity in presence of 1.5 µM NS004. No inhibitory effect was observed at high concentration of NS004 with both wt- and G551D-CFTR. Whole-cell recordings of CFTR chloride currents from cells expressing wild-type or G551D-CFTR in the presence of NS004 were linear, time- and voltage-independent. The inhibitory profile of G551D-CFTR channel activity was similar to that of wild type, i.e., inhibition by glibenclamide (100 µM) and DPC (250 µM) but not by DIDS (200 µM) nor calixarene (100 nM). These results show that NS004 activates wt-CFTR channel and restores G551D-CFTR channel activity, the potency of which depends on both the concentration of NS004 and the phosphorylation status of CFTR.

Inhibition of ATP-sensitive K+ channels by substituted benzo[c]quinolizinium CFTR activators

The substituted benzo[c]quinolizinium compounds MPB-07 and MPB-91 are novel activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. High homologies between CFTR and the sulfonylurea receptor (SUR), which associates with the potassium channel Kir6.2 to form the ATP-sensitive K(+) (K(ATP)) channel, prompted us to examine possible effects of these compounds on K(ATP) channels using electrophysiological recordings and binding assays. Activity of recombinant K(ATP) channels expressed in Xenopus oocytes was recorded in the inside-out configuration of the patch-clamp technique. Channels were practically unaffected by MPB-07 but were fully blocked by MPB-91 with half-inhibition achieved at approximately 20 microM MPB-91. These effects were similar on channels formed by Kir6.2, and either the SUR1 or SUR2A isoforms were independent of the presence of nucleotides. They were not influenced by SUR mutations known to interfere with its nucleotide-binding capacity. MPB-91, but not MPB-07, was able to displace binding of glibenclamide to HEK cells expressing recombinant SUR1/Kir6.2 channels. Glibenclamide binding to native channels from pancreatic MIN6 cells was also displaced by MPB-91. A Kir6.2 mutant able to form channels without SUR was also blocked by MPB-91, but not by MPB-07. These observations demonstrate that neither MPB-07 nor MPB-91 interact with SUR, in spite of its high homology with CFTR, and that MPB-91 blocks K(ATP) channels by binding to the Kir6.2 subunit. Thus, caution should be exercised when planning to use MPB compounds in cystic fibrosis therapy, specially MPB-91 which could nonetheless find interesting applications as the precursor of a new class of K channel blockers.

Genistein Modifies the Activation Kinetics and Magnitude of Phosphorylated Wild-Type and G551D-CFTR Chloride Currents

We have studied the mechanism by which genistein activates cystic fibrosis transmembrane conductance regulator (CFTR) in CHO cells expressing wild type or G551D-CFTR. In wild-type CHO cells, after exposure to 2.5 mM forskolin, 25 mM genistein induced a further 2-fold and rapid increase of the forskolin-activated CFTR current. In both types of cells, when forskolin was added after genistein preincubation, whole-cell current density was greatly reduced compared to that measured when genistein was added after phosphorylation of CFTR, and all activation kinetic parameters were significantly altered. Genistein had no effect on the adenylate cyclase activity. Our results suggest that the occupancy of a putative genistein binding site is critical for the gating mechanism of CFTR chloride channels, which, depending on the phosphorylation status of the R-domain, drives CFTR either into a refractory state or alternatively to a highly activated state.