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Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl− channel

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Original languageEnglish
Pages (from-to)1059–1076
Number of pages18
JournalJournal of Physiology
Volume595
Issue number4
Early online date3 Jan 2017
DOIs
DateAccepted/In press - 21 Oct 2016
DateE-pub ahead of print - 3 Jan 2017
DatePublished (current) - 15 Feb 2017

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl− channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP-binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single-channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open-time constant (τo) of wild-type CFTR. By contrast, the fast closed-time constant (τcf), which describes the short-lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three-state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP-binding site 1, including K464A, D572N and the CF-associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP-dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP-binding site 1 modulates intraburst gating.

    Research areas

  • CFTR, chloride channel, cystic fibrosis

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    Rights statement: This is the author accepted manuscript (AAM). The final published version (version of record) is available online via Wiley at http://onlinelibrary.wiley.com/doi/10.1113/JP273205/abstract. Please refer to any applicable terms of use of the publisher.

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