Mechanism of Glibenclamide Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Cl⁻ Channels Expressed in a Murine Cell Line

Abstract

The sulphonylurea drug glibenclamide is a widely used inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate how glibenclamide inhibits CFTR, we studied CFTR Cl− channels using excised inside‐out membrane patches from cells expressing wild‐type human CFTR. Addition of glibenclamide (10–100 μM) to the intracellular solution caused a concentration‐dependent decrease in the open time of CFTR Cl− channels, but closed times did not change. This suggests that glibenclamide is an open‐channel blocker of CFTR. Glibenclamide is a weak organic acid. Acidification of the intracellular solution relieved glibenclamide inhibition of CFTR, suggesting that the anionic form of glibenclamide inhibits CFTR. To begin to identify the glibenclamide binding site in CFTR, we investigated whether glibenclamide competes with either MgATP or Cl− ions for a common binding site. Glibenclamide inhibition of CFTR was unaffected by nucleotide‐dependent stimulation of CFTR, suggesting that glibenclamide and intracellular MgATP interact with CFTR at distinct sites. Glibenclamide inhibition of CFTR was voltage dependent and enhanced when the external Cl− concentration was decreased. The data suggest that glibenclamide and Cl− ions may compete for a common binding site located within a large intracellular vestibule that is part of the CFTR pore.

Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…4D demonstrates that apparent K d (V) values for CFTR inhibition by OH Ϫ ions are weakly voltage-dependent. The data also indicate that the potency of CFTR inhibition by OH Ϫ ions (apparent K d (0 mV) ϭ 54 Ϯ 6 M; n ϭ 9) approaches that of the best-studied CFTR blocker, the sulfonylurea glibenclamide (K d (0 mV) ϭ 37 M) (20).…”

“…Electrophysiology-CFTR Cl Ϫ channels were recorded in excised inside-out membrane patches using an Axopatch 200A patch-clamp amplifier and pCLAMP software (both from MDS Analytical Technologies, Union City, CA) as described (20).…”

“…To calculated the apparent voltage-dependent dissociation constant (K d ) for OH Ϫ ion inhibition of CFTR and estimate the location of the binding site for OH Ϫ ions within the CFTR pore, we employed the methods described by Sheppard and Robinson (20). For further information, see supplemental "Experimental Procedures.…”

Fluid Secretion Caused by Aerolysin-Like Hemolysin of Aeromonas sobria in the Intestines Is Due to Stimulation of Production of Prostaglandin E 2 via Cyclooxygenase 2 by Intestinal Cells

“…To elucidate whether the CFTR channel, a plasma membrane Cl Ϫ channel, is involved in fluid accumulation caused by hemolysin, the effects of H-89, a cAMP-dependent protein kinase A (PKA) inhibitor (7,27), and glibenclamide, a selective CFTR inhibitor (34,35), were examined. For the control experiment, CT, which causes diarrhea through the activation of CFTR (18,23), was used.…”

Convergence of Pre- and Postsynaptic Influences on Glucosensing Neurons in the Ventromedial Hypothalamic Nucleus

“…Because decreasing extracellular glucose levels should lower intraneuronal ATP levels, such a Cl Ϫ channel should be responsive to changes in the ATP-to-ADP ratio. The cystic fibrosis transmembrane regulator (CFTR) is one such Cl Ϫ conductance, and it is activated by ATP and blocked by sulfonylureas (19). Moreover, CFTR mRNA and protein are expressed in human and rat hypothalamus (20,21).…”