Tolbutamide causes open channel blockade of cystic fibrosis transmembrane conductance regulator Cl- channels

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel that is regulated by protein kinase A and cytosolic nucleotides. Previously, Sheppard and Welsh reported that the sulfonylureas glibenclamide and tolbutamide reduced CFTR whole cell currents. The aim of this study was to quantify the effects of tolbutamide on CFTR gating in excised membrane patches containing multiple channels. We chose tolbutamide because weak (i.e., fast-type) open channel blockers introduce brief events into multichannel recordings that can be readily quantified by current fluctuation analysis. Inspection of current records revealed that the addition of tolbutamide reduced the apparent single-channel current amplitude and increased the open-channel noise, as expected for a fast-type open channel blocker. The apparent decrease in unitary current amplitude provides a measure of open probability within a burst (P0 Burst), and the resulting concentration-response relationship was described by a simple Michaelis-Menten inhibition function. The concentration of tolbutamide causing a 50% reduction of Po Burst (540 +/- 20 microM) was similar to the concentration producing a 50% inhibition of short-circuit current across T84 colonic epithelial cell monolayers (400 +/- 20 microM). Changes in CFTR gating were then quantified by analyzing current fluctuations. Tolbutamide caused a high-frequency Lorentzian (corner frequency, fc > 300 Hz) to appear in the power density spectrum. The fc of this Lorentzian component increased as a linear function of tolbutamide concentration, as expected for a pseudo-first-order open-blocked mechanism and yielded estimates of the on rate (koff = 2.8 +/- 0.3 microM-1 s-1), the off rate (kon = 1210 +/- 225 s-1), and the dissociation constant (KD = 430 +/- 80 microM). Based on these observations, we propose that there is a bimolecular interaction between tolbutamide and CFTR, causing open channel blockade.

Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers

“…Other members of the sulfonylurea family also block the CFTR channel [8,37,41,45]. Therefore, we designed experiments to probe both TM 6 and TM12 alanine mutants by using blockers of this family.…”

“…The hypoglycemic sulfonylureas, including Tolb, Glip, and Glyb, and the non-sulfonylurea hypoglycemic agent Meglitinide, are used clinically to control the release of insulin from pancreatic β-cells by interaction with the sulfonylurea receptor, SUR, a member of the ATPbinding cassette (ABC) transporter superfamily [1,30,34]. Block of CFTR by Glyb [37,40,41,47,50], Tolb [45], and Meglitinide [8] has been described, although the binding sites for these drugs remain to be identified. We have shown previously that Glyb interacts with the CFTR pore in a complicated manner [47,50].…”

Arylaminobenzoate Block of the Cardiac Cyclic AMP-Dependent Chloride Current

“…By lengthening the cardiac action potential duration, future arylaminobenzoate derivatives might represent a new and unique group of class III antiarrhythmic agents that would be effective during sympathetic stimulation. The action of other putative CFTR channel blockers, such as sulfonylureas Venglarik et al, 1996) and clofibric acid analogues (Walsh and Wang, 1996), also will require careful attention.…”

Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

“…To determine the mechanism of this inhibition, the effect of ibuprofen on CFTR Cl Ϫ channels in excised, inside-out patches from mouse L cells recombinantly expressing CFTR (19) was determined. We characterized previously the kinetics of nucleotide-dependent gating (21,22) and sulfonylurea-dependent block of CFTR (23,31) in these cells in detail. The results of one experiment with ibuprofen are shown in Fig.…”