Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation

Abstract

We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC(50) = 85 microM) and activated CFTR in Calu-3 cells (EC(50) = 47 microM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Channel by β-Adrenergic Agonists and Vasoactive Intestinal Peptide in Rat Smooth Muscle Cells and Its Role in Vasorelaxation

“…3C). This pharmacological profile of inhibition is in perfect agreement with that determined for the epithelial CFTR (19,21,24).…”

“…Functional Study of Chloride Channel Activity in SMC-Chloride channel activity was assayed at 37°C by measuring the rate of iodide ( 125 I) efflux as previously described (18,19) and adapted to SMC. All experiments were performed at 37°C.…”

“…Residual radioactivity was extracted with 0.1 N NaOH and determined using a ␥ counter (Cobra II; PerkinElmer Life Sciences). The fraction of initial intracellular 125 I lost during each time point was determined, and time-dependent rates (k ϭ peak rate, min Ϫ1 ) of 125 I efflux were calculated from k ϭ ln( 125 I t1 / 125 I t2 )/(t 1 Ϫ t 2 ) where 125 I t is the intracellular 125 I at time t, and t 1 and t 2 are successive time points (18,19). Curves were constructed by plotting k versus time.…”

“…Therefore, several series of experiments were con- ducted to investigate first whether CFTR is functional as a cAMP-and agonist-dependent chloride channel and, second, since CFTR is present in the concentric layers of smooth muscle within the thickness of rat aorta, whether it could play a role in the vascular reactivity. We first searched for a cAMP-regulated Cl Ϫ channel activity by measuring the rate of iodide ( 125 I) efflux (18,19) from isolated SMC. We performed two series of experiments.…”

“…The peak rate was 0.148 Ϯ 0.009 min Ϫ1 (n ϭ 20) for cells stimulated by cAMP agonists and 0.067 Ϯ 0.005 min Ϫ1 (n ϭ 8) for cells maintained in basal conditions. Three different classes of Cl Ϫ channel inhibitors were used to establish the pharmacological signature of CFTR: glibenclamide and DPC, inhibitors of CFTR channels (23,24), and the TS-TM calix [4]arene derivative, an inhibitor of outwardly rectifying Cl Ϫ channels but not of CFTR (19,21,24). We found that the stimulation of iodide efflux by cAMP agonists with SMC bathed in high K ϩ saline was fully inhibited (p Ͻ 0.001) by 100 M glibenclamide and 500 M DPC but not by 100 nM TS-TM calix [4]arene (Fig.…”