PMID: 26606940

Wei S, Roessler BC, Icyuz M, Chauvet S, Tao B, Hartman JL 4th, Kirk KL
Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.
FASEB J. 2015 Nov 25. pii: fj.15-278382., [PubMed]
Sentences
No. Mutations Sentence Comment
69 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:69:271
status: NEW
view ABCC1 p.Phe565Leu details
ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:69:322
status: NEW
view ABCC1 p.Gly756Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:69:941
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:69:989
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 26606940:69:1085
status: NEW
view ABCC7 p.Phe337Cys details
ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:69:1037
status: NEW
view ABCC7 p.Phe337Leu details
ABCC1 p.Phe565Ser
X
ABCC1 p.Phe565Ser 26606940:69:220
status: NEW
view ABCC1 p.Phe565Ser details
ABCC1 p.Phe565Ala
X
ABCC1 p.Phe565Ala 26606940:69:169
status: NEW
view ABCC1 p.Phe565Ala details
Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
70 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:70:271
status: NEW
view ABCC1 p.Phe565Leu details
ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:70:322
status: NEW
view ABCC1 p.Gly756Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:70:941
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:70:989
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 26606940:70:1085
status: NEW
view ABCC7 p.Phe337Cys details
ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:70:1037
status: NEW
view ABCC7 p.Phe337Leu details
ABCC1 p.Phe565Ser
X
ABCC1 p.Phe565Ser 26606940:70:220
status: NEW
view ABCC1 p.Phe565Ser details
ABCC1 p.Phe565Ala
X
ABCC1 p.Phe565Ala 26606940:70:169
status: NEW
view ABCC1 p.Phe565Ala details
Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
82 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:82:72
status: NEW
view ABCC1 p.Phe565Leu details
We chose to test for GOF effects in these background constructs because F565L was identified as a hit in the aforementioned screen for mutations that suppress defective transport by the Ycf1p Walker B mutant (22). Login to comment
83 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:83:72
status: NEW
view ABCC1 p.Phe565Leu details
We chose to test for GOF effects in these background constructs because F565L was identified as a hit in the aforementioned screen for mutations that suppress defective transport by the Ycf1p Walker B mutant (22). Login to comment
84 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:84:33
status: NEW
view ABCC1 p.Phe565Leu details
As reported previously (22), the F565L mutation partially rescued the cadmium growth phenotype of the NBD1 Walker B construct (D777N). Login to comment
85 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:85:33
status: NEW
view ABCC1 p.Phe565Leu details
As reported previously (22), the F565L mutation partially rescued the cadmium growth phenotype of the NBD1 Walker B construct (D777N). Login to comment
87 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:87:187
status: NEW
view ABCC1 p.Gly756Asp details
For reference, we also introduced each phenylalanine mutation into the WT Ycf1p background and into an NBD1 signature sequence mutant that is expected to have negligible ATPase activity (G756D in Ycf1p) (16, 37, 38). Login to comment
88 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:88:187
status: NEW
view ABCC1 p.Gly756Asp details
For reference, we also introduced each phenylalanine mutation into the WT Ycf1p background and into an NBD1 signature sequence mutant that is expected to have negligible ATPase activity (G756D in Ycf1p) (16, 37, 38). Login to comment
91 ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:91:336
status: NEW
view ABCC7 p.Tyr1219Gly details
We chose this ATP binding mutant of Yor1p for detailed analysis because 1) our earlier results (15) showed that it was possible to rescue its oligomycin growth phenotype by introducing cytosolic GOF mutations that were predicted by our CFTR findings and 2) detailed ATP titrations can be performed for the analogous CFTR A loop mutant (Y1219G) to explore the mechanism underlying such GOF effects (15). Login to comment
92 ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 26606940:92:31
status: NEW
view ABCC7 p.Asp572Asn details
ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:92:336
status: NEW
view ABCC7 p.Tyr1219Gly details
We chose this ATP binding mutant of Yor1p for detailed analysis because 1) our earlier results (15) showed that it was possible to rescue its oligomycin growth phenotype by introducing cytosolic GOF mutations that were predicted by our CFTR findings and 2) detailed ATP titrations can be performed for the analogous CFTR A loop mutant (Y1219G) to explore the mechanism underlying such GOF effects (15). Login to comment
93 ABCC7 p.Asp572Asn
X
ABCC7 p.Asp572Asn 26606940:93:31
status: NEW
view ABCC7 p.Asp572Asn details
The CFTR NBD1 Walker B mutant (D572N) is a severe processing mutant that cannot be so analyzed (39). Login to comment
107 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:107:174
status: NEW
view ABCC1 p.Gly756Asp details
Mutating the conserved extracellular F565 residue partially restores the function of ATP binding mutants of Ycf1p (Y1281G and D777N), but not of a signature sequence mutant (G756D). Login to comment
108 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:108:174
status: NEW
view ABCC1 p.Gly756Asp details
Mutating the conserved extracellular F565 residue partially restores the function of ATP binding mutants of Ycf1p (Y1281G and D777N), but not of a signature sequence mutant (G756D). Login to comment
109 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:109:109
status: NEW
view ABCC1 p.Gly756Asp details
The WT and ATP binding mutations, Y1281G (top panel) and D777N (middle), or the signature sequence mutation, G756D (bottom), are indicated along with the F565 substitutions, introduced alone or in combination. Login to comment
110 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:110:109
status: NEW
view ABCC1 p.Gly756Asp details
ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:110:270
status: NEW
view ABCC1 p.Gly756Asp details
The WT and ATP binding mutations, Y1281G (top panel) and D777N (middle), or the signature sequence mutation, G756D (bottom), are indicated along with the F565 substitutions, introduced alone or in combination. Login to comment
111 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:111:270
status: NEW
view ABCC1 p.Gly756Asp details
The vertical rectangular box indicates spot cultures analyzed in B and C. B) Restoration of Ycf1p function is observed as differential growth associated with F565 substitutions in the context of the Y1281G (top) and D777N (middle) loss of function mutations but not the G756D signature sequence mutation (bottom). Login to comment
113 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:113:103
status: NEW
view ABCC1 p.Gly756Asp details
C) Functional comparison between each single mutant, Ycf1p-Y1281G (top), Ycf1p-D777N (middle) or Ycf1p-G756D (bottom), and the respective A, S, and L substitutions for F565. Login to comment
114 ABCC1 p.Gly756Asp
X
ABCC1 p.Gly756Asp 26606940:114:103
status: NEW
view ABCC1 p.Gly756Asp details
C) Functional comparison between each single mutant, Ycf1p-Y1281G (top), Ycf1p-D777N (middle) or Ycf1p-G756D (bottom), and the respective A, S, and L substitutions for F565. Login to comment
125 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:125:0
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:125:15
status: NEW
view ABCC7 p.Phe337Ala details
F337S-CFTR and F337A-CFTR exhibited robust GOF properties that included 1) substantial currents that remained following the removal of bath ATP with a scavenger (hexokinase/glucose) and subsequent perfusion with an ATP-free solution and 2) strong activation by the poorly hydrolyzable b,g-imidoadenosine 59-triphosphate (AMP-PNP), which is a weak agonist for WT CFTR (example record in Fig. 4A, data summaries in Fig. 4C, D) (4, 43). Login to comment
126 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:126:0
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:126:12
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:126:15
status: NEW
view ABCC7 p.Phe337Ala details
F337S-CFTR and F337A-CFTR exhibited robust GOF properties that included 1) substantial currents that remained following the removal of bath ATP with a scavenger (hexokinase/glucose) and subsequent perfusion with an ATP-free solution and 2) strong activation by the poorly hydrolyzable b,g-imidoadenosine 59-triphosphate (AMP-PNP), which is a weak agonist for WT CFTR (example record in Fig. 4A, data summaries in Fig. 4C, D) (4, 43). Login to comment
127 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:127:12
status: NEW
view ABCC7 p.Phe337Ser details
Because the F337S mutation exhibited strong GOF effects at the macroscopic current level, its Pos under various activation conditions (ATP, no nucleotide, AMP-PNP)wereestimatedforinside-outpatchesthatwere Figure 3. Login to comment
141 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:141:71
status: NEW
view ABCC7 p.Phe337Ser details
A) Macroscopic current record for excised patch containing hundreds of F337S-CFTR channels showing detectable current after ATP removal and strong AMP-PNP activation. Login to comment
142 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:142:71
status: NEW
view ABCC7 p.Phe337Ser details
A) Macroscopic current record for excised patch containing hundreds of F337S-CFTR channels showing detectable current after ATP removal and strong AMP-PNP activation. Login to comment
147 ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:147:92
status: NEW
view ABCC7 p.Phe337Leu details
B) Macroscopic current record for excised patch containing approximately the same number of F337L- CFTR channels showing that this mutation does not increase ATP-free channel activity or subsequent activation by AMP-PNP. Login to comment
148 ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:148:92
status: NEW
view ABCC7 p.Phe337Leu details
B) Macroscopic current record for excised patch containing approximately the same number of F337L- CFTR channels showing that this mutation does not increase ATP-free channel activity or subsequent activation by AMP-PNP. Login to comment
151 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:151:164
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:151:133
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 26606940:151:106
status: NEW
view ABCC7 p.Phe337Cys details
ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:151:79
status: NEW
view ABCC7 p.Phe337Leu details
Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment
152 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:152:26
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:152:164
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:152:16
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:152:133
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 26606940:152:106
status: NEW
view ABCC7 p.Phe337Cys details
ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:152:79
status: NEW
view ABCC7 p.Phe337Leu details
Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment
153 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:153:26
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:153:16
status: NEW
view ABCC7 p.Phe337Ala details
The results for F337A and F337S were significantly different from WT by unpaired Student`s t test (P , 0.05). Login to comment
159 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:159:36
status: NEW
view ABCC7 p.Phe337Ala details
Identical results were obtained for F337A in separate immunoblots (not shown). Login to comment
160 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:160:36
status: NEW
view ABCC7 p.Phe337Ala details
Identical results were obtained for F337A in separate immunoblots (not shown). Login to comment
161 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:161:53
status: NEW
view ABCC7 p.Phe337Ser details
The results in Fig. 5 confirm that the extracellular F337S mutation increased Po especially following ATP removal and the subsequent addition of AMP-PNP. Login to comment
162 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:162:53
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:162:41
status: NEW
view ABCC7 p.Phe337Leu details
The results in Fig. 5 confirm that the extracellular F337S mutation increased Po especially following ATP removal and the subsequent addition of AMP-PNP. Login to comment
163 ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:163:41
status: NEW
view ABCC7 p.Phe337Leu details
Interestingly, the leucine substitution (F337L), which was a strong GOF mutation in Ycf1p but a weaker GOF mutation in Yor1p, had no detectable GOF effect on CFTR channel activity (Fig. 4B-D). Login to comment
165 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:165:18
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:165:99
status: NEW
view ABCC7 p.Tyr1219Gly details
The extracellular F337S substitution enhances the ATP sensitivities of CFTR channels including the Y1219G ATP binding mutant GOF mutations that increase the ligand-free activities of allosteric proteins such as hormone receptors and neurotransmitter-gated channels also reciprocally enhance ligand sensitivity by biasing the equilibrium toward those conformations with the higher ligand affinities (i.e., the activated receptor or open channel) (14, 15, 44-46). Login to comment
166 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:166:18
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:166:89
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:166:99
status: NEW
view ABCC7 p.Tyr1219Gly details
The extracellular F337S substitution enhances the ATP sensitivities of CFTR channels including the Y1219G ATP binding mutant GOF mutations that increase the ligand-free activities of allosteric proteins such as hormone receptors and neurotransmitter-gated channels also reciprocally enhance ligand sensitivity by biasing the equilibrium toward those conformations with the higher ligand affinities (i.e., the activated receptor or open channel) (14, 15, 44-46). Login to comment
167 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:167:89
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:167:241
status: NEW
view ABCC7 p.Tyr1219Gly details
The ATP titration data in Fig. 6 indicate that such reciprocity is also apparent for the F337S mutation. Login to comment
168 ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:168:241
status: NEW
view ABCC7 p.Tyr1219Gly details
This substitution substantially increased the ATP sensitivity of CFTR activation when introduced either into the WT backgroundor intotheNBD2A loop mutantthatlacks the conserved tyrosine that stacks against the adenine ring of ATP at site 2 (Y1219G-CFTR). Login to comment
172 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:172:0
status: NEW
view ABCC7 p.Phe337Ser details
F337S-CFTR channels have increased Pos under all conditions examined. Login to comment
173 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:173:0
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:173:148
status: NEW
view ABCC7 p.Phe337Ser details
F337S-CFTR channels have increased Pos under all conditions examined. Login to comment
174 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:174:148
status: NEW
view ABCC7 p.Phe337Ser details
A) Unitary current recordings at a single holding potential (260 mV; record inverted for presentation) for an excised inside-out patch containing 6 F337S-CFTR channels. Login to comment
178 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:178:220
status: NEW
view ABCC7 p.Phe337Ser details
B) Corresponding unitary current recordings for a patch containing 5 WT-CFTR channels. Conditions and methods were identical to A. Note the different current scales indicating the lower unitary currents exhibited by the F337S mutant. Login to comment
179 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:179:30
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:179:220
status: NEW
view ABCC7 p.Phe337Ser details
B) Corresponding unitary current recordings for a patch containing 5 WT-CFTR channels. Conditions and methods were identical to A. Note the different current scales indicating the lower unitary currents exhibited by the F337S mutant. Login to comment
180 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:180:30
status: NEW
view ABCC7 p.Phe337Ser details
C-E) Mean Pos for WT-CFTR and F337S-CFTR channels estimated for the indicated conditions. Login to comment
181 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:181:35
status: NEW
view ABCC7 p.Phe337Ser details
Numbers (n) are 4 and 5 for WT and F337S, respectively. Login to comment
182 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:182:35
status: NEW
view ABCC7 p.Phe337Ser details
Numbers (n) are 4 and 5 for WT and F337S, respectively. Login to comment
184 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:184:231
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:184:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S substitution markedly increases the activities of CFTR constructs that cannot be activated by ATP We previously observed that cytosolic GOF mutations increased the channel activity of the most common CF regulation mutant G551D-CFTR (14, 15). Login to comment
185 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:185:231
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:185:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S substitution markedly increases the activities of CFTR constructs that cannot be activated by ATP We previously observed that cytosolic GOF mutations increased the channel activity of the most common CF regulation mutant G551D-CFTR (14, 15). Login to comment
186 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:186:0
status: NEW
view ABCC7 p.Gly551Asp details
G551D-CFTR channel activity in excised membrane patches is strongly increased by bath addition of the natural compound curcumin (14, 15, 48), which reveals the presence of gating-defective channels in the patch (Fig. 7A). Login to comment
187 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:187:0
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:187:87
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:187:16
status: NEW
view ABCC7 p.Phe337Ser details
G551D-CFTR channel activity in excised membrane patches is strongly increased by bath addition of the natural compound curcumin (14, 15, 48), which reveals the presence of gating-defective channels in the patch (Fig. 7A). Login to comment
188 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:188:87
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:188:16
status: NEW
view ABCC7 p.Phe337Ser details
Introducing the F337S substitution markedly increased the control currents mediated by G551D-CFTR channels and correspondingly reduced the relative stimulation by curcumin (Fig. 7B-D). Login to comment
189 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:189:24
status: NEW
view ABCC7 p.Phe337Ser details
Figure 8 shows that the F337S substitution also substantially increased the activity of a CFTR truncation mutant that lacks NBD2 (D1198-CFTR). Login to comment
190 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:190:40
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:190:24
status: NEW
view ABCC7 p.Phe337Ser details
Figure 8 shows that the F337S substitution also substantially increased the activity of a CFTR truncation mutant that lacks NBD2 (D1198-CFTR). Login to comment
191 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:191:7
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:191:40
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:191:23
status: NEW
view ABCC7 p.Phe337Ser details
This construct behaves similarly to the G551D CF mutant in that it exhibits very low, ATP-unresponsive currents in excised patches under control conditions but can be stimulated by curcumin (Fig. 8A) (14, 15, 48). Login to comment
192 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:192:7
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:192:23
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:192:25
status: NEW
view ABCC7 p.Phe337Ser details
As for G551D-CFTR, the F337S substitution substantially increased the control currents and correspondingly reduced the relative stimulation by curcumin when introduced into this NBD2 deletion construct (Fig. 8B-D). Login to comment
193 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:193:25
status: NEW
view ABCC7 p.Phe337Ser details
The currents mediated by F337S/D1198-CFTR were insensitive to addition of the ATP scavenger as expected for a construct lacking 1 of the 2 NBDs that dimerize to form the composite ATP binding sites (Fig. 8E). Login to comment
195 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:195:46
status: NEW
view ABCC7 p.Phe337Ser details
The results of Figs. 7 and 8 confirm that the F337S substitution is a bona fide GOF mutation that promotes the unliganded activities of CFTR constructs that cannot be stimulated by ATP. Login to comment
196 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:196:46
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:196:54
status: NEW
view ABCC7 p.Phe337Ser details
The results of Figs. 7 and 8 confirm that the F337S substitution is a bona fide GOF mutation that promotes the unliganded activities of CFTR constructs that cannot be stimulated by ATP. Login to comment
197 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:197:54
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:197:117
status: NEW
view ABCC7 p.Phe337Ser details
PKA sensitivity is also enhanced by the extracellular F337S mutation A GOF mutation that increases unliganded channel opening might also affect the PKA sensitivity of channel activationgiventhatRdomainphosphorylationisrequired to open the channel even in the absence of ATP binding (Fig. 8) (14). Login to comment
198 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:198:117
status: NEW
view ABCC7 p.Phe337Ser details
This prediction is analogous to the effect of a GOF mutation to increase ligand sensitivity, as was verified for the F337S mutant in the ATP titration experiments shown in Fig. 6. Login to comment
200 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:200:37
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:200:99
status: NEW
view ABCC7 p.Lys978Cys details
To test this idea, the extracellular F337S mutation was combined with the previously characterized K978C mutation, which locates tothe cytosolic side below TM9 (14, 15). Login to comment
201 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:201:37
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:201:156
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:201:4
status: NEW
view ABCC7 p.Lys978Cys details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:201:99
status: NEW
view ABCC7 p.Lys978Cys details
To test this idea, the extracellular F337S mutation was combined with the previously characterized K978C mutation, which locates tothe cytosolic side below TM9 (14, 15). Login to comment
202 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:202:156
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:202:4
status: NEW
view ABCC7 p.Lys978Cys details
The K978C substitution increases ATP-free channel activity and enhances the ATP and PKA sensitivities of CFTR activation similar to that shown here for the F337S mutation (14). Login to comment
203 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:203:97
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:203:103
status: NEW
view ABCC7 p.Lys978Cys details
Removing bath ATP in the standard excised macropatch protocol decreased the currents mediated by F337S/K978C-CFTR by less than 20% (Fig. 10A, B). Login to comment
204 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:204:97
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:204:103
status: NEW
view ABCC7 p.Lys978Cys details
Removing bath ATP in the standard excised macropatch protocol decreased the currents mediated by F337S/K978C-CFTR by less than 20% (Fig. 10A, B). Login to comment
206 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:206:17
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:206:23
status: NEW
view ABCC7 p.Lys978Cys details
More strikingly, F337S/K978C-CFTR appeared to be nearly maximally active in the absence of both PKA and ATP (Fig. 10C-F). Login to comment
207 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:207:12
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:207:17
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:207:18
status: NEW
view ABCC7 p.Lys978Cys details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:207:23
status: NEW
view ABCC7 p.Lys978Cys details
More strikingly, F337S/K978C-CFTR appeared to be nearly maximally active in the absence of both PKA and ATP (Fig. 10C-F). Login to comment
208 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:208:12
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:208:18
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:208:120
status: NEW
view ABCC7 p.Tyr1219Gly details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:208:18
status: NEW
view ABCC7 p.Lys978Cys details
Substantial F337S/K978C-CFTR-mediated currents could be detected for macropatches that were excised in the absence of both PKA and ATP in Figure 6. Login to comment
209 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:209:18
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:209:120
status: NEW
view ABCC7 p.Tyr1219Gly details
The extracellular F337S mutation increases the ATP sensitivity of CFTR activation either in the WT background or in the Y1219G ATP binding mutant. Login to comment
213 ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:213:44
status: NEW
view ABCC7 p.Tyr1219Gly details
n = 4 patches for each construct except the Y1219G single mutant (n = 5). Login to comment
214 ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:214:44
status: NEW
view ABCC7 p.Tyr1219Gly details
n = 4 patches for each construct except the Y1219G single mutant (n = 5). Login to comment
216 ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:216:25
status: NEW
view ABCC7 p.Tyr1219Gly details
# P , 0.05 compared with Y1219G by unpaired Student`s t test. Login to comment
217 ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:217:25
status: NEW
view ABCC7 p.Tyr1219Gly details
# P , 0.05 compared with Y1219G by unpaired Student`s t test. Login to comment
219 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:219:45
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:219:51
status: NEW
view ABCC7 p.Lys978Cys details
In support of this interpretation, the Po of F337S/K978C-CFTR estimatedfrom multichannel records in theabsence ofbothPKAandATP(examplerecordinFig.10D)ranged from 0.34 to 0.95 with a mean Po (6SEM) of 0.53 6 0.08 (n = 7 patches;estimated using the conventional Clampfit protocol; see Methods). Login to comment
220 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:220:45
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:220:51
status: NEW
view ABCC7 p.Lys978Cys details
In support of this interpretation, the Po of F337S/K978C-CFTR estimatedfrom multichannel records in theabsence ofbothPKAandATP(examplerecordinFig.10D)ranged from 0.34 to 0.95 with a mean Po (6SEM) of 0.53 6 0.08 (n = 7 patches;estimated using the conventional Clampfit protocol; see Methods). Login to comment
227 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:227:78
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:227:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S mutation increases the activity of the common CF regulation mutant, G551D-CFTR. Login to comment
228 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:228:78
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:228:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S mutation increases the activity of the common CF regulation mutant, G551D-CFTR. Login to comment
229 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:229:33
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:229:64
status: NEW
view ABCC7 p.Phe337Ser details
Macroscopic currents mediated by G551D-CFTR without or with the F337S substitution. Activation conditions were the same as for Fig.4. Login to comment
230 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:230:33
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:230:64
status: NEW
view ABCC7 p.Phe337Ser details
Macroscopic currents mediated by G551D-CFTR without or with the F337S substitution. Activation conditions were the same as for Fig.4. Login to comment
231 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:231:89
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:231:83
status: NEW
view ABCC7 p.Phe337Ser details
Note the much larger control current and lower relative activation by curcumin for F337S/G551D-CFTR. Login to comment
232 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:232:88
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:232:89
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:232:83
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:232:123
status: NEW
view ABCC7 p.Phe337Ser details
Note the much larger control current and lower relative activation by curcumin for F337S/G551D-CFTR. Login to comment
233 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:233:88
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:233:123
status: NEW
view ABCC7 p.Phe337Ser details
C) Scatter plot showing the generally larger macroscopic control currents at 180 mV for G551D-CFTR channels containing the F337S substitution. Login to comment
236 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:236:47
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:236:86
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:236:79
status: NEW
view ABCC7 p.Phe337Ser details
Mean control currents (6SEMs) were as follows: G551D (0.5 6 0.1 pA; n = 5) and F337S/ G551D (115.5 6 59.2; n = 5). Login to comment
237 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:237:47
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:237:86
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:237:79
status: NEW
view ABCC7 p.Phe337Ser details
Mean control currents (6SEMs) were as follows: G551D (0.5 6 0.1 pA; n = 5) and F337S/ G551D (115.5 6 59.2; n = 5). Login to comment
238 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:238:44
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:238:38
status: NEW
view ABCC7 p.Phe337Ser details
The much lower relative activation of F337S/G551D-CFTR is due to its higher control or baseline currents. Login to comment
239 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:239:44
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:239:38
status: NEW
view ABCC7 p.Phe337Ser details
The much lower relative activation of F337S/G551D-CFTR is due to its higher control or baseline currents. Login to comment
244 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:244:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S mutation enhances the ATP-independent activities of channels lacking NBD2. Login to comment
245 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:245:4
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:245:79
status: NEW
view ABCC7 p.Phe337Ser details
The F337S mutation enhances the ATP-independent activities of channels lacking NBD2. Login to comment
246 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:246:7
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:246:79
status: NEW
view ABCC7 p.Phe337Ser details
A, B) Macroscopic currents mediated by D1198-CFTR channels without or with the F337S substitution. Activation conditions and curcumin concentrations were identical to Fig. 7. Login to comment
247 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:247:7
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:247:107
status: NEW
view ABCC7 p.Phe337Ser details
B) The F337S/D1198-CFTR current was inhibited by adding a high concentration of the voltage-dependent CFTR pore blocker, glibenclamide (300 mM). Login to comment
248 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:248:79
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:248:107
status: NEW
view ABCC7 p.Phe337Ser details
C) Scatter plot showing the larger macroscopic control currents at 180 mV for D1198-CFTR channels with the F337S substitution. Login to comment
249 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:249:79
status: NEW
view ABCC7 p.Phe337Ser details
Mean control currents (6SEMs) were as follows: D1198 (2.7 6 2.3 pA; n = 6) and F337S/ D1198 (177.0 6 75.6; n = 7). Login to comment
250 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:250:116
status: NEW
view ABCC7 p.Phe337Ser details
*P , 0.05 by unpaired Student`s t test. E) Macroscopic current record showing that the control currents mediated by F337S/D1198-CFTR are not reduced by scavenging ATP with hexokinase/ glucose (see Fig. 4 legend for hexokinase/glucose concentrations). Login to comment
251 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:251:116
status: NEW
view ABCC7 p.Phe337Ser details
*P , 0.05 by unpaired Student`s t test. E) Macroscopic current record showing that the control currents mediated by F337S/D1198-CFTR are not reduced by scavenging ATP with hexokinase/ glucose (see Fig. 4 legend for hexokinase/glucose concentrations). Login to comment
252 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:252:43
status: NEW
view ABCC7 p.Phe337Ser details
F) Macroscopic current record showing that F337S/D1198-CFTR channel activity is strongly dependent on PKA phosphorylation. Login to comment
253 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:253:43
status: NEW
view ABCC7 p.Phe337Ser details
F) Macroscopic current record showing that F337S/D1198-CFTR channel activity is strongly dependent on PKA phosphorylation. Login to comment
255 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:255:509
status: NEW
view ABCC1 p.Phe565Leu details
Allosteric repair of ATP binding mutants of a long and short MRP The conserved extracellular phenylalanine was chosen for detailed study for 2 reasons: 1) its location at the extracellular ends of the translocations pathways of CFTR and the long and short MRPs permitted us to test for long-range allosteric effects of extracellular perturbations on the apparent ATP occupancies of the cytosolic NBDs of these different transporters (and on the apparent phosphorylation state of the CFTR R domain) and 2) the F565L substitution in Ycf1p (long MRP in yeast) scored as a hit in a previousintragenic screenfor mutations that rescuedpoor growthon cadmium-containing media for yeast expressing the WalkerB mutant, D777N-Ycf1p(22). Login to comment
256 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:256:81
status: NEW
view ABCC1 p.Phe565Leu details
ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:256:509
status: NEW
view ABCC1 p.Phe565Leu details
Allosteric repair of ATP binding mutants of a long and short MRP The conserved extracellular phenylalanine was chosen for detailed study for 2 reasons: 1) its location at the extracellular ends of the translocations pathways of CFTR and the long and short MRPs permitted us to test for long-range allosteric effects of extracellular perturbations on the apparent ATP occupancies of the cytosolic NBDs of these different transporters (and on the apparent phosphorylation state of the CFTR R domain) and 2) the F565L substitution in Ycf1p (long MRP in yeast) scored as a hit in a previousintragenic screenfor mutations that rescuedpoor growthon cadmium-containing media for yeast expressing the WalkerB mutant, D777N-Ycf1p(22). Login to comment
257 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:257:59
status: NEW
view ABCC1 p.Phe565Leu details
ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:257:81
status: NEW
view ABCC1 p.Phe565Leu details
Wereasonedthat the latter result might be related to an allosteric effect of the F565L substitution on ATP occupancy given that the D777N mutation is expected to reduce Mg-ATP binding affinity (15, 22, 36). Login to comment
258 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:258:59
status: NEW
view ABCC1 p.Phe565Leu details
We confirmed this finding and further showed that the same F565L substitution partially rescued the cadmium growth defect exhibited by a second ATP binding mutant, Y1281G-Ycf1p. Login to comment
259 ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:259:89
status: NEW
view ABCC7 p.Tyr1219Gly details
This in- terpretationisconsistentwiththestronginhibitoryeffectof the analogous mutation (Y1219G)on the ATP sensitivity of CFTR channel activation (15, 36), which could be reversed by introducing a second site GOF mutation of the corresponding phenylalanine. Login to comment
260 ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:260:89
status: NEW
view ABCC7 p.Tyr1219Gly details
This in- terpretationisconsistentwiththestronginhibitoryeffectof the analogous mutation (Y1219G)on the ATP sensitivity of CFTR channel activation (15, 36), which could be reversed by introducing a second site GOF mutation of the corresponding phenylalanine. Login to comment
262 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:262:44
status: NEW
view ABCC1 p.Phe565Leu details
In contrast, only the originally identified F565L substitution reproducibly rescued the ATP binding mutants of Ycf1p. Login to comment
263 ABCC1 p.Phe565Leu
X
ABCC1 p.Phe565Leu 26606940:263:44
status: NEW
view ABCC1 p.Phe565Leu details
ABCC1 p.Phe565Ala
X
ABCC1 p.Phe565Ala 26606940:263:34
status: NEW
view ABCC1 p.Phe565Ala details
In contrast, only the originally identified F565L substitution reproducibly rescued the ATP binding mutants of Ycf1p. Login to comment
264 ABCC1 p.Phe565Ala
X
ABCC1 p.Phe565Ala 26606940:264:34
status: NEW
view ABCC1 p.Phe565Ala details
The other tested Ycf1p mutations (F565A, S) exhibited weak to undetectable rescue. Login to comment
267 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:267:18
status: NEW
view ABCC7 p.Phe337Ser details
The extracellular F337S mutation also increases CFTR sensitivity to cytosolic PKA. Login to comment
268 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:268:18
status: NEW
view ABCC7 p.Phe337Ser details
The extracellular F337S mutation also increases CFTR sensitivity to cytosolic PKA. Login to comment
269 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:269:31
status: NEW
view ABCC7 p.Phe337Ser details
C) Mean PKA titration data for F337S-CFTR and WT-CFTR. Login to comment
270 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:270:31
status: NEW
view ABCC7 p.Phe337Ser details
C) Mean PKA titration data for F337S-CFTR and WT-CFTR. Login to comment
275 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:275:4
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:275:10
status: NEW
view ABCC7 p.Lys978Cys details
The F337S/K978C double mutant is nearly fully active in the absence of both exogenous PKA and ATP. Login to comment
276 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:276:4
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:276:187
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:276:10
status: NEW
view ABCC7 p.Lys978Cys details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:276:193
status: NEW
view ABCC7 p.Lys978Cys details
The F337S/K978C double mutant is nearly fully active in the absence of both exogenous PKA and ATP. Login to comment
277 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:277:187
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:277:193
status: NEW
view ABCC7 p.Lys978Cys details
A) Representative macroscopic current record showing that ATP removal by scavenger addition followed by bath perfusion with an ATP-free solution only modestly decreases the activities of F337S/K978C-CFTR channels. Conditions were identical to Fig. 4. Login to comment
279 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:279:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S data set is from Figure 4C. Login to comment
280 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:280:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S data set is from Figure 4C. Login to comment
281 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:281:58
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:281:64
status: NEW
view ABCC7 p.Lys978Cys details
C) Representative macroscopic current record showing that F337S/K978C-CFTR channels are nearly maximally active in excised patches in the absence of both ATP and exogenous PKA. Login to comment
282 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:282:58
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:282:64
status: NEW
view ABCC7 p.Lys978Cys details
C) Representative macroscopic current record showing that F337S/K978C-CFTR channels are nearly maximally active in excised patches in the absence of both ATP and exogenous PKA. Login to comment
283 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:283:93
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:283:99
status: NEW
view ABCC7 p.Lys978Cys details
D) Unitary current recording at a single holding potential for an excised patch containing 3 F337S/K978C-CFTR channels. Login to comment
284 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:284:93
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:284:99
status: NEW
view ABCC7 p.Lys978Cys details
D) Unitary current recording at a single holding potential for an excised patch containing 3 F337S/K978C-CFTR channels. Login to comment
287 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:287:88
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:287:94
status: NEW
view ABCC7 p.Lys978Cys details
E) Gap-free record at a single holding potential for an excised patch containing 80-100 F337S/K978C-CFTR channels showing substantial activity in the absence of bath PKA and ATP. Login to comment
288 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:288:29
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:288:88
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:288:35
status: NEW
view ABCC7 p.Lys978Cys details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:288:94
status: NEW
view ABCC7 p.Lys978Cys details
E) Gap-free record at a single holding potential for an excised patch containing 80-100 F337S/K978C-CFTR channels showing substantial activity in the absence of bath PKA and ATP. Login to comment
289 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:289:29
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:289:35
status: NEW
view ABCC7 p.Lys978Cys details
F) Stationary noise plot for F337S/K978C-CFTR channels in the absence of PKA and ATP. Login to comment
299 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:299:140
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:299:150
status: NEW
view ABCC7 p.Phe337Ala details
The present results confirmed these earlier findings and also revealed that a subset of F337 substitutions are strong GOF mutants, notably, F337S and F337A. Login to comment
300 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:300:357
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:300:140
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 26606940:300:150
status: NEW
view ABCC7 p.Phe337Ala details
The present results confirmed these earlier findings and also revealed that a subset of F337 substitutions are strong GOF mutants, notably, F337S and F337A. Login to comment
301 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 26606940:301:357
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:301:4
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:301:143
status: NEW
view ABCC7 p.Tyr1219Gly details
GOF effects on CFTR channel gating were operationally defined as 1) large fractional currents that persist following ATP removal, 2) robust activation by the normally weak agonist, AMP-PNP, and 3) substantial increases in channel activities when introduced into CFTR constructs that cannot be activated by ATP, namely, the most common CF regulation mutant (G551D-CFTR) or a truncation mutant lacking NBD2 (D1198-CFTR). Login to comment
302 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:302:4
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Tyr1219Gly
X
ABCC7 p.Tyr1219Gly 26606940:302:143
status: NEW
view ABCC7 p.Tyr1219Gly details
The F337S substitution also increased the ATP sensitivity of CFTR channel activation when introduced either into the WT background or into the Y1219G-CFTR ATP binding mutant. Login to comment
307 ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:307:4
status: NEW
view ABCC7 p.Phe337Leu details
The F337L mutation did not obviously disrupt CFTR protein maturation or inhibit the control ATP-dependent currents in macropatches (Fig. 4). Login to comment
308 ABCC7 p.Phe337Leu
X
ABCC7 p.Phe337Leu 26606940:308:4
status: NEW
view ABCC7 p.Phe337Leu details
The F337L mutation did not obviously disrupt CFTR protein maturation or inhibit the control ATP-dependent currents in macropatches (Fig. 4). Login to comment
312 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:312:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S GOF mutation also provides insights into how PKA phosphorylation regulates CFTR PKA-mediated phosphorylation is essential for WT CFTR channel activity (9). Login to comment
313 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:313:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S GOF mutation also provides insights into how PKA phosphorylation regulates CFTR PKA-mediated phosphorylation is essential for WT CFTR channel activity (9). Login to comment
314 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:314:38
status: NEW
view ABCC7 p.Phe337Ser details
Two findings from our analysis of the F337S GOF mutant impact our understanding of how PKA regulates CFTR channel activity. Login to comment
315 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:315:38
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:315:64
status: NEW
view ABCC7 p.Phe337Ser details
Two findings from our analysis of the F337S GOF mutant impact our understanding of how PKA regulates CFTR channel activity. Login to comment
316 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:316:64
status: NEW
view ABCC7 p.Phe337Ser details
First, the strong PKA dependence of the channel activity of the F337S/D1198-CFTR truncation construct (Fig. 8F) argues for a regulatory mechanism that is independent of any effect that phosphorylation might have on NBD dimerization (see also 14). Login to comment
318 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:318:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S/ D1198-CFTR construct lacks NBD2, cannot form an NBD1-NBD2 dimer and is unresponsive to ATP. Login to comment
319 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:319:4
status: NEW
view ABCC7 p.Phe337Ser details
The F337S/ D1198-CFTR construct lacks NBD2, cannot form an NBD1-NBD2 dimer and is unresponsive to ATP. Login to comment
323 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:323:140
status: NEW
view ABCC7 p.Phe337Ser details
The second finding that impacts our understanding of the PKA regulatory mechanism was the long-range allosteric effect of the extracellular F337S mutation on PKA sensitivity (Fig. 9). Login to comment
324 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:324:140
status: NEW
view ABCC7 p.Phe337Ser details
The second finding that impacts our understanding of the PKA regulatory mechanism was the long-range allosteric effect of the extracellular F337S mutation on PKA sensitivity (Fig. 9). Login to comment
328 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:328:35
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:328:41
status: NEW
view ABCC7 p.Lys978Cys details
Engineering a superactive CFTR The F337S/K978C double mutant has the highest single-channel activity in the absence of exogenous PKA and ATP of any CFTR construct that we have characterized to date. Login to comment
329 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:329:35
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:329:41
status: NEW
view ABCC7 p.Lys978Cys details
Engineering a superactive CFTR The F337S/K978C double mutant has the highest single-channel activity in the absence of exogenous PKA and ATP of any CFTR construct that we have characterized to date. Login to comment
331 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:331:24
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:331:34
status: NEW
view ABCC7 p.Lys978Cys details
We anticipated that the F337S and K978C mutations would have additive GOF effects on ATP-free CFTR activity because they locate to opposite sides of the pore where they presumably impact CFTR structure in different ways. Login to comment
332 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 26606940:332:24
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys978Cys
X
ABCC7 p.Lys978Cys 26606940:332:34
status: NEW
view ABCC7 p.Lys978Cys details
We anticipated that the F337S and K978C mutations would have additive GOF effects on ATP-free CFTR activity because they locate to opposite sides of the pore where they presumably impact CFTR structure in different ways. Login to comment