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 EC 50 ≈ 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 EC 50 ≈ 1.5 µM. We also determined the half-maximal effective concentration of forskolin (EC 50 ≈ 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.
The Journal of Membrane Biology – Springer Journals
Published: Sep 18, 2003
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