Direct Comparison of NPPB and DPC as Probes of CFTR Expressed in Xenopus Oocytes |
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Authors: | Z-R Zhang S Zeltwanger NA McCarty |
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Institution: | (1) Departments of Physiology and Pediatrics, Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, GA 30322, USA, US |
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Abstract: | Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We
have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB
and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel
currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at −100 mV. The affinity for
NPPB calculated from single-channel block, K
D
= 35 μm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type
(WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single
binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its
effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB.
We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects
(i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate
that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for
binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes
for overlapping regions in the pore.
Received: 14 October 1999/Revised: 18 January 2000 |
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Keywords: | : Cystic fibrosis — Cystic fibrosis transmembrane conductance regulator — Chloride channel blocker — Anion channel — Arylaminobenzoate |
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