Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.

Abstract

The gene defective in cystic fibrosis encodes a C1-channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 v,M, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses ~ 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 -+ 0.4 pS in symmetric 150 mM CI-. A subconductance state, measuring ~60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 tzM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at I'm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (~1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.

Polystyrene nanoparticles activate ion transport in human airway epithelial cells

“…The subsequent addition of forskolin, an activator of adenylyl cyclase (10 µM), further increased the short-circuit current by 15.8 ± 2.9 µA/cm 2 (plateau I sc , Figure 2A) and this effect was blocked by diphenylamine-2-carboxylate, a blocker of CFTR (1 mM). 22 In the absence of nanoparticles, forskolin activated the short-circuit current by 16.2 ± 1.0 µA/cm 2 (plateau I sc , Figure 2B). However, the subsequent addition of N20 after forskolin resulted in a significantly lower short-circuit current response (4.7 ± 2.4 µA/cm 2 ) when compared with control responses (P , 0.05, n = 3, Figure 2B), showing that there is in fact an overlap between the mechanism of short-circuit current activation by N20 and forskolin.…”

Inhibition of CFTR channels by a peptide toxin of scorpion venom

“…Oocytes were prepared for study by manually removing the vitelline membrane after shrinking in hypertonic solution (34). Pipettes were pulled in four stages from borosilicate glass (Sutter) and fire polished.…”

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl– currents

“…The large Cl -currents in the presence of GST-syn 1A∆C were not affected by DIDS, which blocks a number of Cl -channels other than CFTR (26). Conversely, diphenylamine carboxylate (DPC), which does block CFTR Cl -channels (27), completely inhibited the currents that were potentiated by GST-syn 1A∆C ( Figure 5). The I-V relationship in the presence of either reagent was also approximately linear, which is a hallmark of CFTR Cl -channels (28).…”