Devra P. Rich

Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel

CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in cAMP-responsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR Cl- channel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is sufficient for regulation of Cl- channel activity.

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.

Cystic fibrosis transmembrane conductance regulator : a chloride channel with novel regulation

Michael J. Welsh,* Matthew P. Anderson,* Devra P. Rich,* Herbert A. Berger,* Gerene M. Denning,* Lynda S. Ostedgaard,* David N. Sheppard,* Seng H. Cheng,+ Richard J. Gregory,+ and Alan E. Smith+ *Howard Hughes Medical Institute Department of internal Medicine Department of Physiology and Biophysics University of Iowa College of Medicine Iowa City, Iowa 52242 +Genzyme Corporation Framingham, Massachusetts 01701

Identification of revertants for the cystic fibrosis ΔF508 mutation using STE6-CFTR chimeras in yeast

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis; the most common mutation is deletion of phenylalanine at position 508 (delta F508). We constructed STE6-CFTR chimeras with portions of the first nucleotide-binding domain (NBD1) of the yeast STE6 a-factor transporter replaced by portions of CFTR NBD1. The chimeras were functional in yeast, but mating efficiency decreased when delta F508 was introduced into NBD1. We isolated two delta F508 revertant mutations (R553M and R553Q) that restored mating; both were located within the CFTR NBD1 sequence. Introduction of these revertant mutations into human CFTR partially corrected the processing and Cl- channel gating defects caused by the delta F508 mutation. These results suggest that the NBD1s of CFTR and STE6 share a similar structure and function and that, in CFTR, the regions containing F508 and R553 interact. They also indicate that the abnormal conformation produced by delta F508 can be partially corrected by additional alterations in the protein.

The amino-terminal portion of CFTR forms a regulated CI− channel

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel consists of two motifs (each containing a membrane-spanning domain [MSD] and a nucleotide-binding domain [NBD]) linked by an R domain. We tested the hypothesis that one MSD-NBD motif could form a Cl- channel. The amino-terminal portion of CFTR (D836X, which contains MSD1, NBD1, and the R domain) formed Cl- channels with conductive properties identical to those of CFTR. However, channel regulation differed. Although phosphorylation increased activity, channels opened without phosphorylation. MgATP stimulated D836X more potently than CFTR and may interact at more than one site. These data and migration of D836X on sucrose density gradients suggest that D836X may function as a multimer. Thus, the amino-terminal portion of CFTR contains all of the structures required to build a regulated Cl- channel.

Chapter 7 The CFTR Chloride Channel

Publisher Summary Cystic fibrosis transmembrane conductance regulator (CFTR) appears to be the first identified member of a novel class of Cl channels. CFTR is a regulated Cl − channel located in the apical membrane of several Cl − secretory epithelia. Regulation of CFTR is complex and involves both phosphorylation and an interaction with intracellular nucleotides. The combination of a molecular biologic approach (using site-directed mutagenesis and expression of CFTR in heterologous cells), a biochemical approach (using CFTR-specific antibodies), and an electrophysiologic approach (using the patch-clamp technique) is beginning to provide essential information about the structure and function of the various domains of this interesting channel. CFTR is regulated by phosphorylation of the R domain. First, addition of cyclic adenosine monophosphate (cAMP) agonists increases the apical membrane Cl − permeability of normal, but not CF, epithelia, and addition of cAMP agonists activates CFTR Cl − channels in heterologous cells, expressing recombinant CFTR. The biophysical properties of recombinant CFTR Cl − channels are the same as those of endogenous CFTR Cl − channels. Under baseline conditions, there is little, if any, Cl − current in cells endogenously, expressing CFTR. Following the addition of cAMP agonists, there is a dramatic increase in Cl − current in both whole-cell patch-clamp studies in individual epithelial cells. Cl − channel activation does not occur in response to an increase in intracellular Ca 2+ .

Function and Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator

Electrolyte transport across airway epithelia controls the quantity and composition of respiratory tract fluid. In patients with cystic fibrosis (CF), cAMP-stimulated Cl− secretion by airway epithelia is defective. This defect results from a failure of cAMP to activate apical membrane Cl− channels (Quinton, 1990).