Activating Cystic Fibrosis Transmembrane Conductance Regulator Channels with Pore Blocker Analogs

Abstract

Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is ⌬F508-CFTR, which inefficiently traffics to the surfaces of most cells. The ⌬F508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and ⌬F508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment ⌬F508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and ⌬F508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of ⌬F508-CFTR channels in excised membrane patches by 10 -15-fold under conditions in which wildtype channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the ⌬F508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

“…Thus, the interaction of Lys-946 with the COMMD1 protein (32) or Asp-979 (33) may also result , and S768C/S832C/V510A (C) by using a ramp protocol (Ϯ80 mV). NPPB-AM is a CFTR-specific opener (25). D, fractional inhibition of the current by diamide for three CFTR mutants (n ϭ 3-5; *, p Ͻ 0.05, unpaired t test); error bars, S.E.…”

An Electrostatic Interaction at the Tetrahelix Bundle Promotes Phosphorylation-dependent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Opening

“…Cell Culture, DNA Constructs, and Transfections-HEK 293T cells were cultured in Dulbecco's modified Eagle's Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS) and transiently transfected with wild type (WT) or mutant CFTR cDNA as described previously (12,28). All point mutations and deletion mutations were generated using appropriate mutagenic oligonucleotides, verified by DNA sequencing and subcloned into the pCDNA3 mammalian expression vector (Invitrogen).…”

“…Electrophysiology and Data Analysis-Macroscopic and unitary currents were recorded in the excised, inside-out configuration using previously described patch clamp techniques (12,28). Patch pipettes were pulled from Corning 8161 glass to a tip resistance of 1.5-2.0 M⍀ for macroscopic recordings and 10 -12 M⍀ for unitary current recordings.…”

“…All point mutations and deletion mutations were generated using appropriate mutagenic oligonucleotides, verified by DNA sequencing and subcloned into the pCDNA3 mammalian expression vector (Invitrogen). Cells were transfected as described previously (12,28) and grown for 24 -72 h at 37°C in DMEM plus 10% FBS without antibiotics prior to experimentation.…”

“…2A shows a typical macroscopic current record for an excised patch containing many wild type (WT) CFTR channels (Ͼ1000) that were activated by normally saturating concentrations of MgATP and PKA. Inhibiting the PKA in the bath by adding protein kinase A inhibitory peptide (PKI) resulted in a partial deactivation of the macroscopic current due presumably to the activity of membrane associate phosphatases in the membrane patch (12,28). Subsequent addition of compounds that were previously shown to potentiate wild type and mutant CFTR activity (NPPB-AM and curcumin, Ref.…”