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Voltage-dependent Block of the Cystic Fibrosis Transmembrane Conductance Regulator Cl- Channel by Two Closely Related Arylaminobenzoates

McCarthy, N. A. and McDonough, S. and Cohen, B. N. and Riordan, J. R. and Davidson, N. and Lester, H. A. (1993) Voltage-dependent Block of the Cystic Fibrosis Transmembrane Conductance Regulator Cl- Channel by Two Closely Related Arylaminobenzoates. Journal of General Physiology, 102 (1). pp. 1-23. ISSN 0022-1295. PMCID PMC2229162. doi:10.1085/jgp.102.1.1.

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The gene defective in cystic fibrosis encodes a Cl- 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 µM, 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 Cl^-. 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 µM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at V_m =-100 mV and not at V_m = +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 V_m = -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.

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Lester, H. A.0000-0002-5470-5255
Additional Information:© 1993 Rockefeller University Press. Original version received 7January 1993 and accepted version received 17 March 1993. Published July 1, 1993. The authors wish to thank Dr. Xian-cheng Yang for advice on single-channel analysis, Prof. Anita Zimmerman and Prof. Dennis Dougherty for helpful discussions, Dr. Michael Quick for assistance, and Dr. B. K. Kobilka and colleagues (Dept. of Cellular and Molecular Physiology, Stanford University Medical Center, Stanford, CA) for supplying us with the β2-AR cDNA. This project was supported by grants from the Cystic Fibrosis Foundation (Z139) and from the NIH (GM-29836). N. A. McCarty was supported by an NIH NRSA fellowship (DK08559) and S. McDonough was supported by an NIH training grant (GM-07737).
Funding AgencyGrant Number
Cystic Fibrosis FoundationZ139
NIH Predoctoral FellowshipDK08559
Issue or Number:1
PubMed Central ID:PMC2229162
Record Number:CaltechAUTHORS:20120309-113518954
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Official Citation:Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates. N A McCarty, S McDonough, B N Cohen, J R Riordan, N Davidson, and H A Lester 102:1-23. doi:10.1085/jgp.102.1.1
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:29664
Deposited By: Ruth Sustaita
Deposited On:12 Mar 2012 15:49
Last Modified:09 Nov 2021 19:12

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