ABCMdb

PMID: 9813087

Jiang Q, Mak D, Devidas S, Schwiebert EM, Bragin A, Zhang Y, Skach WR, Guggino WB, Foskett JK, Engelhardt JF
Cystic fibrosis transmembrane conductance regulator-associated ATP release is controlled by a chloride sensor.
J Cell Biol. 1998 Nov 2;143(3):645-57., 1998-11-02 [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu ABCC7 p.Arg347Glu Despite the lack of a need for Cl-conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl ϾϾ Br; R347P, Cl ϾϾ Br; R347E, Br ϾϾ Cl; R334W, Cl ϭ Br). Login to comment 80 ABCC7 p.Arg347Glu The cAMP-stimulated Cl- and Br- conductances of the R347E mutant (5.3 Ϯ 2.1 ␮s and 12.15 Ϯ 3.3 ␮s, respectively) were much lower than that of the wild type. Login to comment 89 ABCC7 p.Arg347Glu For mutant R347, a cDNA segment was cut out from the PTM1-R347E CFTR construct (kindly provided by Dr. M. Welsh, University of Iowa) by restriction enzymes Erev,Br Erev,Cl RT F ------- PBr Br- [ ] PCl Cl- [ ] --------------------------       ln=- MroI and Bst1107I, and was subcloned into the pBQ-CFTR plasmid between the same restriction sites. Login to comment 90 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro Mutants R334W and R347P were constructed by replacing a SpH I-Xba I segment in the PSP-CFTR with a corresponding segment cut out from mutants PTM-R334W and PTM-R347P (provided by Dr. M.J. Welsh; Sheppard et al., 1993), respectively. Login to comment 92 ABCC7 p.Lys370* The COOH-terminal truncation mutant, TMD1 CFTR, was constructed by introducing a stop codon at K370X followed by an EcoRV restriction site using the mutagenic oligonucleotide 5Ј-GCAATAAACTAAATACAG- GATATCTTAC-3Ј. Login to comment 93 ABCC7 p.Met265Val The NH2-terminal truncation mutant ⌬259-M265V CFTR was constructed as previously described (Piazza Carroll et al., 1995). Login to comment 157 ABCC7 p.Met265Val ABCC7 p.Met265Val Dissociation of Cl-Conductance and ATP Efflux Functions Associated with CFTR Because the results indicated that CFTR was necessary but not sufficient for ATP permeability, we attempted to identify the important domains in CFTR by comparing wild-type CFTR with two deletion mutants, TMD1 and ⌬259-M265V. Login to comment 158 ABCC7 p.Met265Val The TMD1 mutant encompassing the NH2-terminal portion of CFTR encoded the first 369 amino acids (first six transmembrane helices), while in the ⌬259-M265V mutant the first 259 amino acids were deleted and the methionine 265 was mutated to a valine. Login to comment 161 ABCC7 p.Met265Val In contrast, expression of the ⌬259-M265V mutant was associated with near wild-type rates of ATP efflux in the absence of detectable Cl-conductances (Fig. 3). Login to comment 195 ABCC7 p.Met265Val Comparison of CFTR-modulated ATP release between wild-type, TMD1, and ⌬259-M265V CFTR cRNA-injected Xenopus oocytes. Login to comment 200 ABCC7 p.Met265Val An equal number (N) of wt CFTR, TMD1, and ⌬259-M265V cRNA-injected oocytes were assessed for ATP efflux within each of two batches of oocytes. Login to comment 206 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu Mutations in the CFTR Channel Pore Alter the Halide Dependence of ATP Release To explore the mechanisms by which changes in the extracellular Cl-concentration affect activation of ATP release, we examined the CFTR mutants R334W, R347P, and R347E. Login to comment 208 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu Immunoprecipitation studies using a rabbit antisera raised against a synthetic peptide corresponding to residues 45-65 in the CFTR NH2 terminus demonstrated similar levels of protein expression in wtCFTR, R334W, R347P, and R347E cRNA-injected oocytes (Fig. 6 A). Login to comment 215 ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro The stimulated Cl-conductance of R347P was comparable to that of the wtCFTR (⌬GcAMP ϭ 125 Ϯ 28 ␮s). Login to comment 216 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Glu ABCC7 p.Arg347Glu In contrast, the stimulated Cl-conductances were 4and 20-fold lower in the R334W and R347E mutants, respectively (Fig. 6 and Table II). Login to comment 218 ABCC7 p.Arg334Trp The mutants R347P and R334W demonstrated slight alterations in their conductance ratios (GBr/GCl; Br- Ͼ Cl- ) as compared with wtCFTR (Cl ഡ Br). Login to comment 242 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu The halide dependence of CFTR-modulated ATP release was similarly analyzed in R334W, R347P, and R347E cRNA-injected oocytes. Login to comment 244 ABCC7 p.Arg347Pro The halide dependence of ATP release for R347P CFTR was similar to that of wtCFTR (Cl- ϾϾ Br- ); JATP in the presence of Cl- (2.0 Ϯ 0.98 pmoles/min) was eightfold greater than that in the presence of Br- (0.26 Ϯ 0.17 pmoles/min; Fig. 7 and Table II). Login to comment 246 ABCC7 p.Arg334Trp In contrast, the R334W mutant had no halide dependence in the activation of ATP release (JATP[Cl] ϭ 3.4 Ϯ 1.3 pmoles/min, JATP [Br] ϭ 3.7 Ϯ 1.5 pmoles/min; Fig. 7). Login to comment 247 ABCC7 p.Arg347Glu Most importantly, the halide dependence in the R347E mutant was reversed to Br- ϾϾ Cl- ; JATP in the presence of Cl- (1.5 Ϯ 0.49 pmoles/min) was 7.3-fold lower than that in the presence of Br- (11 Ϯ 4.8 pmoles/min; Fig. 7 and Table II). Login to comment 248 ABCC7 p.Arg347Glu Interestingly, exposure of R347E-expressing oocytes to extracellular Br- had a lasting affect on JATP, even after extracellular Br- was replaced with Cl- . Login to comment 274 ABCC7 p.Met265Val However, the CFTR mutant ⌬259-M265V, which failed to conduct Cl- , also conferred near wild-type rates of ATP release. Login to comment 277 ABCC7 p.Met265Val First, the CFTR mutant ⌬259-M265V demonstrated near wild-type rates of CFTR-modulated ATP release despite its inability to conduct Cl- . Login to comment 279 ABCC7 p.Arg347Glu ABCC7 p.Arg347Glu Furthermore, the R347E had a greater than 20-fold reduced GBr as compared with wtCFTR, yet the magnitude of JATP in the presence of extracellular Br- was sixfold higher for R347E when compared with wtCFTR cRNA-injected oocytes. Login to comment 295 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu Mutants of CFTR that alter charged arginine residues within the channel pore including R334W, R347E, and R347P were used. Login to comment 298 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu The Wc I/V relationships for wtCFTR, R347P, R347E, R334W cRNA, and water-injected oocytes are given in B. Login to comment 303 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu These results depict average I/V relationships from N experiments for wtCFTR (N ϭ 5), R347E (N ϭ 5), R347P (N ϭ 5), R334W (N ϭ 8), and water (N ϭ 7)-injected oocytes from at least two independent batches of oocytes. Login to comment 307 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu We therefore characterized the effects of arginine mutations R334W, R347P, and R347E on CFTR-modulated ATP release. Login to comment 314 ABCC7 p.Arg347Glu In contrast, the R347E mutation caused pronounced alterations of both electrophysiologic as well as ATP release properties of CFTR. Login to comment 321 ABCC7 p.Arg334Trp ABCC7 p.Arg334Glu The number of oocytes (N) compiled for each mutant are given with the number of independent experiments for wtCFTR ϭ 3, R347P ϭ 2, R334E ϭ 3, R334W ϭ 2, and water ϭ 3. Login to comment 326 ABCC7 p.Arg334Trp tant R334W demonstrated no halide dependence in the ATP efflux rates (JATP[Br]/JATP[Cl] ϭ 1.1) when compared with wtCFTR, suggesting that multiple residues within the CFTR channel pore may be involved in halide binding and subsequent conformational changes necessary for activation of CFTR-modulated ATP release. Login to comment 338 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu Summary of Electrophysiologic Measurements on CFTR Mutants Wild type R347P R347E R334W Mock ⌬GCl (cAMP) (␮s) 121 Ϯ 35 125 Ϯ 28 5.3 Ϯ 2.1 32 Ϯ 20 -0.61 Ϯ 0.62 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) (N ϭ 7) GBr /GCl 0.93 Ϯ 0.01 1.12 Ϯ 0.01 2.36 Ϯ 0.23 1.12 Ϯ 0.03 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) - PBr /PCl 1.13 Ϯ 0.02 1.02 Ϯ 0.01 1.00 Ϯ 0.04 1.22 Ϯ 0.02 (N ϭ 5) (N ϭ 5) (N ϭ 5) (N ϭ 8) - JATP (Cl) 8.7 Ϯ 3.4 2.0 Ϯ 0.98 1.5 Ϯ 0.49 3.4 Ϯ 1.3 0.021 Ϯ 0.001 (N ϭ 15) (N ϭ 9) (N ϭ 11) (N ϭ 7) (N ϭ 15) JATP (Br) 1.9 Ϯ 0.51 0.26 Ϯ 0.17 11.0 Ϯ 4.8 3.7 Ϯ 1.5 0.013 Ϯ 0.006 (N ϭ 15) (N ϭ 9) (N ϭ 11) (N ϭ 7) (N ϭ 15) JATP (Br)/JATP (Cl) 0.22 0.13 7.3 1.1 - CFTR cRNAs were injected into Xenopus oocytes and evaluated for both electrophysiologic and ATP release characteristics. Login to comment 354 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Glu We thank Dr. Welsh for his mutant CFTR cDNAs R334W, R347E, and R347P, and his thoughtful review of this manuscript. Login to comment