ABCMdb

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 ABCC1 p.Gly756Asp ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Cys ABCC7 p.Phe337Leu ABCC1 p.Phe565Ser ABCC1 p.Phe565Ala 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 ABCC1 p.Gly756Asp ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Cys ABCC7 p.Phe337Leu ABCC1 p.Phe565Ser ABCC1 p.Phe565Ala 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 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 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 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 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 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 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 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 ABCC7 p.Tyr1219Gly 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 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 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 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 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 ABCC1 p.Gly756Asp 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 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 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 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 ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 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 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 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 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 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 ABCC7 p.Phe337Ala ABCC7 p.Phe337Cys ABCC7 p.Phe337Leu 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Cys ABCC7 p.Phe337Leu 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 ABCC7 p.Phe337Ala 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 Identical results were obtained for F337A in separate immunoblots (not shown). Login to comment 160 ABCC7 p.Phe337Ala Identical results were obtained for F337A in separate immunoblots (not shown). Login to comment 161 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Leu 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 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 ABCC7 p.Tyr1219Gly 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 ABCC7 p.Phe337Ser ABCC7 p.Tyr1219Gly 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 ABCC7 p.Tyr1219Gly 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 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 F337S-CFTR channels have increased Pos under all conditions examined. Login to comment 173 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser F337S-CFTR channels have increased Pos under all conditions examined. Login to comment 174 ABCC7 p.Phe337Ser 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 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 ABCC7 p.Phe337Ser 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 C-E) Mean Pos for WT-CFTR and F337S-CFTR channels estimated for the indicated conditions. Login to comment 181 ABCC7 p.Phe337Ser Numbers (n) are 4 and 5 for WT and F337S, respectively. Login to comment 182 ABCC7 p.Phe337Ser Numbers (n) are 4 and 5 for WT and F337S, respectively. Login to comment 184 ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 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 ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser 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 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 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Phe337Ser ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Phe337Ser ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys 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 ABCC7 p.Phe337Ser ABCC7 p.Tyr1219Gly ABCC7 p.Lys978Cys 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 ABCC7 p.Tyr1219Gly 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 n = 4 patches for each construct except the Y1219G single mutant (n = 5). Login to comment 214 ABCC7 p.Tyr1219Gly n = 4 patches for each construct except the Y1219G single mutant (n = 5). Login to comment 216 ABCC7 p.Tyr1219Gly # P , 0.05 compared with Y1219G by unpaired Student`s t test. Login to comment 217 ABCC7 p.Tyr1219Gly # P , 0.05 compared with Y1219G by unpaired Student`s t test. Login to comment 219 ABCC7 p.Phe337Ser ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Phe337Ser The F337S mutation increases the activity of the common CF regulation mutant, G551D-CFTR. Login to comment 228 ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser The F337S mutation increases the activity of the common CF regulation mutant, G551D-CFTR. Login to comment 229 ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser Note the much larger control current and lower relative activation by curcumin for F337S/G551D-CFTR. Login to comment 232 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser Note the much larger control current and lower relative activation by curcumin for F337S/G551D-CFTR. Login to comment 233 ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser 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 ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser 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 ABCC7 p.Gly551Asp ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 The F337S mutation enhances the ATP-independent activities of channels lacking NBD2. Login to comment 245 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser The F337S mutation enhances the ATP-independent activities of channels lacking NBD2. Login to comment 246 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 ABCC7 p.Phe337Ser 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 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 *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 *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 F) Macroscopic current record showing that F337S/D1198-CFTR channel activity is strongly dependent on PKA phosphorylation. Login to comment 253 ABCC7 p.Phe337Ser F) Macroscopic current record showing that F337S/D1198-CFTR channel activity is strongly dependent on PKA phosphorylation. Login to comment 255 ABCC1 p.Phe565Leu 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 ABCC1 p.Phe565Leu 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 ABCC1 p.Phe565Leu 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 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 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 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 In contrast, only the originally identified F565L substitution reproducibly rescued the ATP binding mutants of Ycf1p. Login to comment 263 ABCC1 p.Phe565Leu ABCC1 p.Phe565Ala In contrast, only the originally identified F565L substitution reproducibly rescued the ATP binding mutants of Ycf1p. Login to comment 264 ABCC1 p.Phe565Ala The other tested Ycf1p mutations (F565A, S) exhibited weak to undetectable rescue. Login to comment 267 ABCC7 p.Phe337Ser The extracellular F337S mutation also increases CFTR sensitivity to cytosolic PKA. Login to comment 268 ABCC7 p.Phe337Ser The extracellular F337S mutation also increases CFTR sensitivity to cytosolic PKA. Login to comment 269 ABCC7 p.Phe337Ser C) Mean PKA titration data for F337S-CFTR and WT-CFTR. Login to comment 270 ABCC7 p.Phe337Ser C) Mean PKA titration data for F337S-CFTR and WT-CFTR. Login to comment 275 ABCC7 p.Phe337Ser ABCC7 p.Lys978Cys 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 ABCC7 p.Phe337Ser ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 The F337S data set is from Figure 4C. Login to comment 280 ABCC7 p.Phe337Ser The F337S data set is from Figure 4C. Login to comment 281 ABCC7 p.Phe337Ser ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Phe337Ser ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys F) Stationary noise plot for F337S/K978C-CFTR channels in the absence of PKA and ATP. Login to comment 299 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ser ABCC7 p.Tyr1219Gly 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 ABCC7 p.Tyr1219Gly 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 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 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 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 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 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 ABCC7 p.Phe337Ser 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 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 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 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 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 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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 ABCC7 p.Lys978Cys 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