ABCMdb

PMID: 7690753

Rich DP, Berger HA, Cheng SH, Travis SM, Saxena M, Smith AE, Welsh MJ
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.
J Biol Chem. 1993 Sep 25;268(27):20259-67., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala The additional sites are likelylocated within the R domain: CFTRAR-S660A, which lacksmuch of the R domain (residues 708-835)and replaces Ser-660 with an alanine,was no longer regulated byPKA. Login to comment 8 ABCC7 p.Ser660Ala The additional sites are likelylocated within the R domain: CFTRAR-S660A, which lacksmuch of the R domain (residues 708-835)and replaces Ser-660 withan alanine,was no longer regulated byPKA. Login to comment 47 ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala Mutants are named by the number of the amino acid residue preceded by the wild-type amino acid and followed bythe aminoacid to which the residue was changed using the single-letter amino acid code; for example,inthemutant CFTR S66OA,S737A,S795A,S813A (also called CFTR S-Quad-A for convenience), alanines are substituted for serines at amino acid positions 660, 737, 795, and 813. Login to comment 48 ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala Mutants are named by the number of the amino acid residue preceded by the wild-type amino acid and followed bythe aminoacid to which the residue was changed using the single-letter aminoacid code; for example,inthemutant CFTR S66OA,S737A,S795A,S813A (also called CFTR S-Quad-A for convenience), alanines are substituted for serines at amino acid positions 660, 737, 795, and 813. Login to comment 65 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser813Ala ABCC7 p.Ser686Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser700Ala In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) were transiently expressed in HeLa cells. Login to comment 66 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser813Ala ABCC7 p.Ser686Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser700Ala In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) weretran- siently expressed in HeLa cells. Login to comment 89 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser813Ala Functional assay of CFTR S-Quad-Aby SPQ fluorescence.The changein SPQfluorescenceis shown for HeLa cellsexpress- ing wild-type CFTR (n = 53, where n = number of cells) or CFTR S-Quad-A tS660A,S737A,S795A,S813A)(n = 53) and for virus only- infectedcells(no plasmid) as a negative control (n = 47). Login to comment 94 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala Single-channelanalysis of gle-channel traces from an excised, in-cell expressing CFTR S-Quad-A side-out membrane patch from a HeLa (S660A,S737A,S795A,S813A).Each trace is about 14s long. Login to comment 95 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala Single-channelanalysis of gle-channel traces from an excised, in-cell expressing CFTR S-Quad-A side-out membrane patch from a HeLa (S660A,S737A,S795A,S813A).Each trace is about 14s long. Login to comment 109 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala RESULTS Serine-to-Alanine Substitutionsin the R Domain Do NotAbolish CFTR Cl- Channel Activity-& a firststep to address the possibility that additional PKA phosphorylation sites might regulate CFTR, we asked whether the S-Quad-A (S660A,S737A,S795A,S813A) mutant could still bephosphorylated in vivofollowing CAMPstimulation. Login to comment 111 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala RESULTS Serine-to-Alanine Substitutions in the R Domain Do NotAbolish CFTR Cl-Channel Activity-& a firststep to address the possibility that additional PKA phosphorylation sites might regulate CFTR, we asked whether the S-Quad-A (S660A,S737A,S795A,S813A) mutant could still be phosphorylated in vivofollowing CAMPstimulation. Login to comment 121 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp ABCC7 p.Ser686Asp ABCC7 p.Ser795Asp ABCC7 p.Ser660Asp ABCC7 p.Ser712Asp ABCC7 p.Ser700Asp ABCC7 p.Ser813Asp The single-channel open-stateprobabil- ity for wild-type CFTR (n = 14), CFTR S-Quad-A (S600A,S737A, S712A,S737A,S768A,S795A,S813A) (n = 7), or CFTR S-Oct-D (S660D,S686D,S700D,S712D,S737D,S768D,S795D,S813D)(n = 7in ATP alone (-PKA);n = 10 inPKA and ATP (+PkX))C1-channels was determined as described under "Experimental Procedures." Login to comment 123 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser813Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp ABCC7 p.Ser686Asp ABCC7 p.Ser795Asp ABCC7 p.Ser660Asp ABCC7 p.Ser712Asp ABCC7 p.Ser700Asp ABCC7 p.Ser813Asp S795A,S813A) (n = 5). Login to comment 124 ABCC7 p.Ser660Ala ABCC7 p.Ser686Ala ABCC7 p.Ser700Ala CFTR S-Oct-A (S660A,S686A,S700A, CFTR A 795 737 4+B5 -813 B," -02 -B3 sion was high, we expected the mutant channels to be less active than wild-type CFTR C1-channels. Login to comment 125 ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala S795A,S813A) (n = 5). Login to comment 126 ABCC7 p.Ser660Ala ABCC7 p.Ser686Ala ABCC7 p.Ser700Ala CFTR S-Oct-A (S660A,S686A,S700A, CFTR A 795 737 4+B5 - 8 1 3 B," -02 -B3 sion was high, we expected the mutant channels to be less active than wild-type CFTR C1-channels. Login to comment 139 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala CFTR (A) or CFTR S-Quad-A (S660A,S737A,S795A,S813A)( B )were transiently expressed inCOS-7 cells. Login to comment 142 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala CFTR (A) or CFTR S-Quad-A (S660A,S737A,S795A,S813A) ( B )were transiently expressed inCOS-7 cells. Login to comment 161 ABCC7 p.Ser660Ala CFTRAR-S660A is similar toCFTRAR except that Ser-660, the sole remaining in vivo PKA phosphorylation site (13), is mutatedto alanine. Login to comment 165 ABCC7 p.Ser660Ala CFTRAR-S660A is similar toCFTRAR except that Ser-660, the sole remaining in vivo PKA phosphorylation site (13), is mutatedto alanine. Login to comment 166 ABCC7 p.Ser660Ala CFTRAR-S660A C1-channels had biophysical properties similar towild-type CFTR C1-channels: single-channel slope conductance was 9.2 *0.4 pS (n = 3).The poof CFTRARS660ACl- channels was0.15 *0.04 (n= 6) in the presence ofATPalone and did not increasefollowingaddition of PKA (n = 4) (Fig. lOB). Login to comment 170 ABCC7 p.Ser660Ala CFTRAR-S660A C1-channels had biophysical properties similar towild-type CFTR C1-channels: single-channel slope conductance was 9.2 *0.4 pS (n = 3).The poof CFTRARS660ACl- channels was 0.15 *0.04 (n= 6) in the presence ofATPalone and did not increasefollowingaddition of PKA (n = 4) (Fig. lOB). Login to comment 174 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser686Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser700Ala Fig. 1lA shows that simultaneous substitutionof the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A MockS-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B)or pMT-CFl`R S-Oct-A FIG.9. Login to comment 176 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser686Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser700Ala COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A)(C). Login to comment 178 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser686Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser700Ala Fig. 1lA shows that simultaneous substitution of the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A Mock S-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B) or pMT-CFl`R S-Oct-A FIG. 9. Login to comment 180 ABCC7 p.Ser660Ala ABCC7 p.Ser737Ala ABCC7 p.Ser795Ala ABCC7 p.Ser813Ala ABCC7 p.Ser686Ala ABCC7 p.Ser768Ala ABCC7 p.Ser712Ala ABCC7 p.Ser700Ala COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) (C). Login to comment 205 ABCC7 p.Ser737Asp ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp ABCC7 p.Ser686Asp ABCC7 p.Ser795Asp ABCC7 p.Ser795Asp ABCC7 p.Ser660Asp ABCC7 p.Ser712Asp ABCC7 p.Ser700Asp ABCC7 p.Ser813Asp ABCC7 p.Ser813Asp Functional analysisof serine-to-aspartate mutantsof CFTR.A, the changes in SPQ fluorescence ofHeLa cells expressing wild-type CFTR (n = 56), CFTR S-Quad-D (S600D,S737D, S795D,S813D) (n = 24), CFTR S-Hex-D (S660D,S686D,S700D, S712D,S737D,S768D,S795D,S813D)( n = 23), or virus only-infected control cells (n = 53) after substitution of NO; for Iat 0 min. Login to comment 209 ABCC7 p.Ser737Asp ABCC7 p.Ser737Asp ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.Ser686Asp ABCC7 p.Ser686Asp ABCC7 p.Ser795Asp ABCC7 p.Ser795Asp ABCC7 p.Ser795Asp ABCC7 p.Ser660Asp ABCC7 p.Ser660Asp ABCC7 p.Ser712Asp ABCC7 p.Ser712Asp ABCC7 p.Ser700Asp ABCC7 p.Ser700Asp ABCC7 p.Ser813Asp ABCC7 p.Ser813Asp ABCC7 p.Ser813Asp B, time course of current changes inan excised, inside-outmembrane patchfrom a HeLa cell expressingCFTR S-Oct-D (S660D,S686D,S700D,S712D,S737D,S768D,S795D,S813D) C1- chan- nels.ATP (0.88 mM)and PKA (75 m)were added tothe cytosolic(bath) side of the membrane as indicated by the burs. Login to comment 213 ABCC7 p.Ser737Asp ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp ABCC7 p.Ser686Asp ABCC7 p.Ser686Asp ABCC7 p.Ser795Asp ABCC7 p.Ser795Asp ABCC7 p.Ser660Asp ABCC7 p.Ser660Asp ABCC7 p.Ser712Asp ABCC7 p.Ser700Asp ABCC7 p.Ser700Asp ABCC7 p.Ser813Asp ABCC7 p.Ser813Asp S737D,S795D,S813D) (n = 37), CFTR S-Oct-D (S660D,S686D,S700D, vitro or in vivo (Figs.6 and 9). Login to comment 217 ABCC7 p.Ser737Asp ABCC7 p.Ser686Asp ABCC7 p.Ser795Asp ABCC7 p.Ser660Asp ABCC7 p.Ser700Asp ABCC7 p.Ser813Asp S737D,S795D,S813D) (n = 37), CFTR S-Oct-D (S660D,S686D,S700D, vitro or in vivo (Figs.6 and 9). Login to comment 232 ABCC7 p.Ser660Ala CFTRAR-S660A contains only one "nonclassic" (R-X-X-Sor R-X-S) PKA phosphorylation site (Ser-753)in thedeleted segment; its consensus sequence is R-I-S.Alternatively, if PKAphosphorylated "nonclassic"sites within other partsof CFTR which interacted with theR domain, then deletion of part of the R domainwould disrupt those interactions whether or not the additional sites were phosphorylated. Login to comment 234 ABCC7 p.Ser660Ala Finally, we cannot rule out thepossi- bility that PKA simply cannot interact with CFTRAR-S660A, even though it does interact withCFTRAR (14). Login to comment 235 ABCC7 p.Ser660Ala CFTRAR-S660A contains only one "nonclassic" (R-X-X-Sor R-X-S) PKA phosphorylation site (Ser-753)in thedeleted segment; its consensus sequence is R-I-S.Alternatively, if PKAphosphorylated "nonclassic"sites within other parts of CFTR which interacted with theR domain, then deletion of part of the R domainwould disrupt those interactions whether or not the additional sites were phosphorylated. Login to comment 237 ABCC7 p.Ser660Ala Finally, we cannot rule out thepossibility that PKA simply cannot interact with CFTRAR-S660A, even though it does interact withCFTRAR (14). Login to comment