The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing

Monika Kloch, Michał Milewski, Ewa Nurowska, Beata Dworakowska, Garry R Cutting, Krzysztof Dołowy

Research output: Contribution to journalArticle

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR→AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR→AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

Original languageEnglish (US)
Pages (from-to)169-180
Number of pages12
JournalCellular Physiology and Biochemistry
Volume25
Issue number2-3
DOIs
StatePublished - 2010

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Cystic Fibrosis Transmembrane Conductance Regulator
Chloride Channels
Nucleotides
Adenosine Triphosphate
Hydrolysis
ATP-Binding Cassette Transporters
Dipeptides
HEK293 Cells
Histidine
Alanine
Carrier Proteins

Keywords

  • ABC transporters
  • CFTR
  • Cystic fibrosis
  • H-loop
  • Nucleotide-binding domain

ASJC Scopus subject areas

  • Physiology

Cite this

The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing. / Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R; Dołowy, Krzysztof.

In: Cellular Physiology and Biochemistry, Vol. 25, No. 2-3, 2010, p. 169-180.

Research output: Contribution to journalArticle

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