Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 2927K) PPT slides of all figures Supplemental Material Index Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JGP Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Csanády, L. Articles by Gadsby, D. C. Search for Related Content PubMed PubMed Citation Articles by Csanády, L. Articles by Gadsby, D. C. Social Bookmarking                      What's this?

¹ Department of Medical Biochemistry, Semmelweis University, 1085 Budapest, Hungary
² Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
³ Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10021
⁴ Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021

Correspondence to David C. Gadsby: gadsby{at}rockefeller.edu

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P_o) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P_o-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

Key Words: ABC transporters • chloride ion-channel gating • multiple phosphorylation sites • mass spectrometry • autoradiography

K.W. Chan's present address is Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106.

J. Qin's present address is Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030.

D.T. McLachlin's present address is BioMedCom Consultants Inc., Montreal, Canada.

Abbreviations used in this paper: ABC, ATP-binding casette; CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide binding domain; R, regulatory; WT, wild type.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. M. Paumi, M. Chuk, I. Chevelev, I. Stagljar, and S. Michaelis Negative Regulation of the Yeast ABC Transporter Ycf1p by Phosphorylation within Its N-terminal Extension J. Biol. Chem., October 3, 2008; 283(40): 27079 - 27088. [Abstract] [Full Text] [PDF]

				T.-Y. Chen and T.-C. Hwang CLC-0 and CFTR: Chloride Channels Evolved From Transporters Physiol Rev, April 1, 2008; 88(2): 351 - 387. [Abstract] [Full Text] [PDF]

				P. Artigas, S. J. Al'Aref, E. A. Hobart, L. F. Diaz, M. Sakaguchi, S. Straw, and O. S. Andersen 2,3-Butanedione Monoxime Affects Cystic Fibrosis Transmembrane Conductance Regulator Channel Function through Phosphorylation-Dependent and Phosphorylation-Independent Mechanisms: The Role of Bilayer Material Properties Mol. Pharmacol., December 1, 2006; 70(6): 2015 - 2026. [Abstract] [Full Text] [PDF]

				L. Csanady, A. C. Nairn, and D. C. Gadsby Thermodynamics of CFTR Channel Gating: A Spreading Conformational Change Initiates an Irreversible Gating Cycle J. Gen. Physiol., November 1, 2006; 128(5): 523 - 533. [Abstract] [Full Text] [PDF]

				S. G. Bompadre, T. Ai, J. H. Cho, X. Wang, Y. Sohma, M. Li, and T.-C. Hwang CFTR Gating I: Characterization of the ATP-dependent Gating of a Phosphorylation-independent CFTR Channel ({Delta}R-CFTR) J. Gen. Physiol., March 28, 2005; 125(4): 361 - 375. [Abstract] [Full Text] [PDF]

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents