CFTR gating I

Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (ΔR-CFTR)

Silvia G. Bompadre, Tomohiko Ai, Jeong Han Cho, Xiaohui Wang, Yoshiro Sohma, Min Li, Tzyh Chang Hwang

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed ΔR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the ΔR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal ΔR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of ΔR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and ΔR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the ΔR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

Original languageEnglish (US)
Pages (from-to)361-375
Number of pages15
JournalJournal of General Physiology
Volume125
Issue number4
DOIs
StatePublished - Apr 2005
Externally publishedYes

Fingerprint

Adenosine Diphosphate
Adenosine Triphosphate
Phosphorylation
Chloride Channels
Cricetulus
Phosphoric Monoester Hydrolases
Serine
Ovary
Catalytic Domain
Hydrolysis
Nucleotides
Binding Sites
Membranes

Keywords

  • ABC transporter
  • Chloride channel
  • Gating mode
  • Phosphorylation
  • Single-channel kinetics

ASJC Scopus subject areas

  • Physiology

Cite this

CFTR gating I : Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (ΔR-CFTR). / Bompadre, Silvia G.; Ai, Tomohiko; Cho, Jeong Han; Wang, Xiaohui; Sohma, Yoshiro; Li, Min; Hwang, Tzyh Chang.

In: Journal of General Physiology, Vol. 125, No. 4, 04.2005, p. 361-375.

Research output: Contribution to journalArticle

Bompadre, Silvia G. ; Ai, Tomohiko ; Cho, Jeong Han ; Wang, Xiaohui ; Sohma, Yoshiro ; Li, Min ; Hwang, Tzyh Chang. / CFTR gating I : Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (ΔR-CFTR). In: Journal of General Physiology. 2005 ; Vol. 125, No. 4. pp. 361-375.
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T2 - Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (ΔR-CFTR)

AU - Bompadre, Silvia G.

AU - Ai, Tomohiko

AU - Cho, Jeong Han

AU - Wang, Xiaohui

AU - Sohma, Yoshiro

AU - Li, Min

AU - Hwang, Tzyh Chang

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AB - The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed ΔR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the ΔR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal ΔR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of ΔR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and ΔR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the ΔR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

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KW - Chloride channel

KW - Gating mode

KW - Phosphorylation

KW - Single-channel kinetics

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