ABCMdb

PMID: 11124965

Kogan I, Ramjeesingh M, Huan LJ, Wang Y, Bear CE
Perturbation of the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits its atpase activity.
J Biol Chem. 2001 Apr 13;276(15):11575-81. Epub 2000 Dec 21., 2001-04-13 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Arg347Asp Mutations of residues lining the channel pore (e.g. R347D) are typically thought to cause disease by altering the interaction of Cl- with the pore. Login to comment 5 ABCC7 p.Arg347Asp However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. Login to comment 7 ABCC7 p.Arg347Asp Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. Login to comment 20 ABCC7 p.Gly551Asp Moreover, the disease-causing mutation in the conserved Walker C motif of the first NBD of CFTR, i.e. CFTR- G551D, impaired both ATPase activity and the rate of channel opening (18). Login to comment 102 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp The Pore Mutant CFTR-R347D Exhibits Altered ATPase Activity-Direct evidence for communication between the chloride channel pore and the catalytic domains of CFTR came from assessments of the ATPase activity of a disease-causing variant of CFTR bearing an amino acid substitution (Arg to Asp) at position 347 within the putative pore region. Login to comment 105 ABCC7 p.Arg347Asp However, the specific activity of PKA-phosphorylated CFTR-R347D was FIG. 1. Login to comment 125 ABCC7 p.Arg347Asp The increase in ATPase activity associated with PKA-treated CFTR-R347D samples was not due to PKA itself, since treatment with this enzyme conferred less than 8% of the total activity measured for the phosphorylated mutant (see legend to Fig. 5A for details). Login to comment 127 ABCC7 p.Arg347Asp Whereas the Vmax of phosphorylated wild type CFTR protein is about 50 nmol/mg/min (18), the Vmax determined for phosphorylated R347D protein is 1.1 nmol/mg/min (Fig. 5B, Table I). Login to comment 128 ABCC7 p.Gly551Asp This reduced ATPase activity is comparable with that obtained for mutations of conserved residues thought to reside in the nucleotide binding pocket in the NBDs (e.g. G551D; (18)). Login to comment 129 ABCC7 p.Arg347Asp Defective ATPase activity by CFTR-R347D mutant was not due to FIG. 4. Login to comment 140 ABCC7 p.Arg347Asp Characterization of the effect of CFTR-R347D mutation on the ATPase activity. Login to comment 142 ABCC7 p.Arg347Asp The mean Ϯ S.E. is shown for nine phosphorylated and nonphosphorylated wild type CFTR preparations. For CFTR-R347D preparations, each bar represents the activity of duplicate values. Login to comment 144 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp PKA phosphorylation of liposomes alone, treated in the same manner as CFTR-R347D preparations, accounted for less than 8% of the ATPase activity observed for the phosphorylated CFTR-R347D protein (0.16 nmol of ATP hydrolyzed/2 h versus 2.03 nmol of ATP hydrolyzed/2 h). Login to comment 146 ABCC7 p.Arg347Asp Panel B, MgATP dependence of the catalytic activity of purified and either phosphorylated or nonphosphorylated CFTR-R347D protein. Login to comment 149 ABCC7 p.Arg347Asp Each sample contained ϳ8 ␮g of purified, reconstituted CFTR-R347D protein, and duplicate or triplicate samples were assessed. Login to comment 150 ABCC7 p.Arg347Asp Panel C, effect of ionic strength on the ATPase activity of phosphorylated wild type CFTR and CFTR-R347D proteins. Login to comment 152 ABCC7 p.Arg347Asp Duplicate samples of wild type protein and triplicate samples of CFTR-R347D were studied. Login to comment 153 ABCC7 p.Arg347Asp global misfolding of the nucleotide binding folds, as the apparent affinity of the phosphorylated mutant for MgATP was comparable, even somewhat higher, than previously reported for phosphorylated wild type protein (Km ϭ 0.1 mM for CFTR-R347D versus Km ϭ 0.3 mM for wild type protein, respectively (18)). Login to comment 154 ABCC7 p.Arg347Asp The pore blocker DPC exerted a similar inhibitory effect on the ATPase activity of phosphorylated CFTR-R347D, as it did on the ATPase activity of wild type protein (67.3 Ϯ 6.1% of control versus 66.8 Ϯ 3.6% of control, respectively). Login to comment 155 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp These results support previous reports suggesting that DPC interacts with a pore-lining residue other than Arg-347, probably Ser341 (44), and that the R347D mutation does not disrupt DPC binding to this site by inducing global misfolding. Login to comment 156 ABCC7 p.Arg347Asp Moreover, a similar inhibitory trend was observed for increasing salt concentration on the ATPase activity of R347D protein (Fig. 5C), as compared with the wild type protein. Login to comment 167 ABCC7 p.Arg347Asp Finally, we observed a significant difference in the inhibitory effect of GSH (10 mM) on the ATPase activity of wild type and CFTR-R347D proteins, 39 and 63% of untreated controls, respectively (Fig. 6C, p ϭ 0.04). Login to comment 176 ABCC7 p.Arg117His ABCC7 p.Ser1118Phe With regards to CFTR, there is indirect evidence supporting interaction between permeation and gating, in that certain mutations in the transmembrane segments of CFTR, namely S1118F in TM11 (33) and the disease-causing mutant R117H (50), exhibit altered channel open times. Login to comment 188 ABCC7 p.Ser341Ala ABCC7 p.Thr1134Phe Such detailed molecular mapping studies have been initiated by McCarty and co-workers (52) in studies of the voltage-dependent block by DPC and NPPB in wild type and mutant (S341A and T1134F) CFTR. Login to comment 190 ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp Certain organic anions including GSH, GSSG, and gluconate have been previously shown to block chloride ion flux through TABLE I Kinetic parameters of PKA-phosphorylated and non-phosphorylated wild-type and R347D proteins Km Vmax ϩPKA -PKA ϩPKA -PKA mM nmol/mg/min Wild typea 0.3 1.0 54 51 R347D 0.1 1.1 1.1 0.2 a Data for wild type CFTR were reported in our previous studies (18). Login to comment 194 ABCC7 p.Arg347Asp We found further evidence for direct communication between the pore domain and NBDs of CFTR when investigating the catalytic activity of CFTR-R347D, a disease-causing variant with a mutation in TM6 associated with mild disease (45). Login to comment 195 ABCC7 p.Arg347Asp The R347D mutation led to inhibition of the ATPase activity of CFTR, suggesting that the region in which this arginine resides participates in the physical communication between the pore and the NBDs. This residue is thought to reside in the inner vestibule of the CFTR channel and has been implicated in anion binding within the pore (45, 50). Login to comment 198 ABCC7 p.Arg347Asp Both the interaction of DPC with the pore and mutation of the putative pore-lining residue, R347D, induced similar changes in the catalytic activity of phosphorylated CFTR, namely, both of these perturbations caused a 3-5-fold decrease in the Vmax of the enzyme. Login to comment 211 ABCC7 p.Arg347Asp Second, mutation of the pore-lining residue R347D led to a change in the extent of blockade of CFTR ATPase activity by GSH. Login to comment 221 ABCC7 p.Arg347Asp Effect of glutathione on the catalytic activity of wild type and CFTR-R347D proteins. Login to comment 228 ABCC7 p.Arg347Asp Panel C, effect of 10 mM GSH on the catalytic activity of phosphorylated wild type (WT, open) and CFTR-R347D (hatched) proteins relative to control (no GSH). Login to comment 236 ABCC7 p.Arg347Asp Acknowledgments-We thank Dr. Mary Corey (Research Institute, Hospital for Sick Children) for the assistance with the statistical analysis and Dr. Johanna Rommens for providing us with cDNA coding for the mutant R347D (Research Institute, Hospital for Sick Children). Login to comment