VOLTAGE-DEPENDENT BLOCK OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CL- CHANNEL BY 2 CLOSELY-RELATED ARYLAMINOBENZOATES

The gene defective in cystic fibrosis encodes a Cl- channel, the cysti c 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 X enopus oocytes by DPC or by a closely related molecule, flufenamic aci d (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by F FA, both at 200 muM, requires several minutes to reach full effect. Bl ockade is voltage dependent, suggesting open-channel block: currents a t positive potentials are not affected but currents at negative potent ials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the insi de. In single-channel recordings from excised patches without blockers , the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subcon ductance state, measuring approximately 60% of the main conductance, w as often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hype rpolarizing voltages, bursts were interrupted by brief closures. Eithe r DPC or FFA (50 muM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at V(m) = - 100 mV and n ot at V(m) = + 100 mV, in agreement with whole-cell experiments. DPC i nduced a higher frequency of flickers from the cytoplasmic side than t he extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at - 100 mV with application from either side. In cell-attached patch recordings with D PC or FFA applied to the bath, there was flickery block at V(m) = - 10 0 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a sing le 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-d irected mutagenesis to describe the permeation pathway.