ABCMdb

PMID: 24876383

Wei S, Roessler BC, Chauvet S, Guo J, Hartman JL 4th, Kirk KL
Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.
J Biol Chem. 2014 Jul 18;289(29):19942-57. doi: 10.1074/jbc.M114.562116. Epub 2014 May 29., [PubMed]

Sentences

No. Mutations Sentence Comment

108 ABCC7 p.Lys978Cys Fig. 1A also shows the location of a previously characterized GOF mutation (K978C) that locates near the base of TM9 in the outer TM collar (16). Login to comment 109 ABCC7 p.Lys978Cys Fig. 1A also shows the location of a previously characterized GOF mutation (K978C) that locates near the base of TM9 in the outer TM collar (16). Login to comment 135 ABCC7 p.Pro355Ala C-E, mean single channel open probabilities afe; S.E. (error bars) for WT (n afd; 6 patches) and P355A-CFTR channels (n afd; 7) under the indicated conditions. Login to comment 136 ABCC7 p.Pro355Ala C-E, mean single channel open probabilities afe; S.E. (error bars) for WT (n afd; 6 patches) and P355A-CFTR channels (n afd; 7) under the indicated conditions. Login to comment 143 ABCC7 p.Gly551Asp ABCC7 p.Pro355Ala The P355A Mutation Rescues Channels That Are Normally ATP-unresponsive-We next determined whether a Pro-355 GOF mutation could promote the activities of channels that normally cannot be stimulated by ATP, namely a truncation mutant lacking NBD2 (èc;1198-CFTR) and an NBD1 signature sequence mutant (G551D-CFTR). Login to comment 144 ABCC7 p.Gly551Asp ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala The P355A Mutation Rescues Channels That Are Normally ATP-unresponsive-We next determined whether a Pro-355 GOF mutation could promote the activities of channels that normally cannot be stimulated by ATP, namely a truncation mutant lacking NBD2 (èc;1198-CFTR) and an NBD1 signature sequence mutant (G551D-CFTR). Login to comment 145 ABCC7 p.Pro355Ala When combined with the P355A substitution, these constructs also expressed sufficiently well for patch clamp analysis, although, like the single mutants, their expression was lower than wild type CFTR (see immunoblot in Fig. 4G). Login to comment 147 ABCC7 p.Pro355Ala Fig. 4, B-D, shows that introducing the P355A mutation into the èc;1198-CFTR background created channels with FIGURE 3. Login to comment 148 ABCC7 p.Pro355Ala Fig. 4, B-D, shows that introducing the P355A mutation into the èc;1198-CFTR background created channels with FIGURE 3. Login to comment 157 ABCC7 p.Pro355Ser ABCC7 p.Pro355Ala ABCC7 p.Pro355Phe Mean percentages of ATP-free currents afe; S.E. (error bars) were as follows: WT, 0.7 afe; 0.2% (n afd; 5); P355A, 5.0 afe; 1.4% (n afd; 10); P355S, 7.3 afe; 2.0% (n afd; 7); and P355F, 5.4 afe; 1.5% (n afd; 5). Login to comment 158 ABCC7 p.Pro355Ser ABCC7 p.Pro355Ala ABCC7 p.Pro355Phe Mean percentages of ATP-free currents afe; S.E. (error bars) were as follows: WT, 0.7 afe; 0.2% (n afd; 5); P355A, 5.0 afe; 1.4% (n afd; 10); P355S, 7.3 afe; 2.0% (n afd; 7); and P355F, 5.4 afe; 1.5% (n afd; 5). Login to comment 159 ABCC7 p.Pro355Ala C, macroscopic current record showing substantial activation of P355A-CFTR by 2 mM AMP-PNP when added following ATP removal. Login to comment 160 ABCC7 p.Pro355Ala C, macroscopic current record showing substantial activation of P355A-CFTR by 2 mM AMP-PNP when added following ATP removal. Login to comment 164 ABCC7 p.Pro355Ser ABCC7 p.Pro355Ala ABCC7 p.Pro355Phe TABLE 1 Single channel data for multiple Pro-355 CFTR mutants in the absence of ATP and following AMP-PNP activation Conditions were identical to those described in the legend to Fig. 2. n afd; 6 (WT), 7 (P355A), 8 (P355F), and 5 (P355S) patches, respectively. Login to comment 165 ABCC7 p.Pro355Ser ABCC7 p.Pro355Ser ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala ABCC7 p.Pro355Phe ABCC7 p.Pro355Phe TABLE 1 Single channel data for multiple Pro-355 CFTR mutants in the absence of ATP and following AMP-PNP activation Conditions were identical to those described in the legend to Fig. 2. n afd; 6 (WT), 7 (P355A), 8 (P355F), and 5 (P355S) patches, respectively. Login to comment 166 ABCC7 p.Pro355Ser ABCC7 p.Pro355Ala ABCC7 p.Pro355Phe WT P355A P355F P355S Mean Po With ATP 0.3074afe;.00514 0.4078 afe; 0.0601 0.4867 afe; 0.0429 0.5212 afe; 0.1048 Without ATP 0.0009 afe; 0.0004 0.0217 afe; 0.0065a 0.0413 afe; 0.0145a 0.0365 afe; 0.0131a With AMP-PNP 0.0127 afe; 0.0114 0.1942 afe; 0.0619a 0.1112 afe; 0.0342a 0.1549 afe; 0.0651a Mean open frequency (sd1a;1 ) With ATP 0.222 afe; 0.021 0.270 afe; 0.028 0.384 afe; 0.049 0.334 afe; 0.059 Without ATP 0.004 afe; 0.001 0.020 afe; 0.002a 0.046 afe; 0.010a 0.028 afe; 0.006a With AMP-PNP 0.013 afe; 0.006 0.131 afe; 0.053a 0.104 afe; 0.035a 0.118 afe; 0.045a a p b0d; 0.05 compared with WT. Login to comment 167 ABCC7 p.Gly551Asp ABCC7 p.Pro355Ala The P355A mutation also partially rescued the activity of G551D-CFTR, a severely defective gating mutant that is common in the CF patient population (Fig. 4, E and F). Login to comment 168 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Pro355Ala The P355A mutation also partially rescued the activity of G551D-CFTR, a severely defective gating mutant that is common in the CF patient population (Fig. 4, E and F). Login to comment 169 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The G551D mutation disrupts the NBD1 signature sequence that is essential for ATP-dependent gating and strongly inhibits ATP hydrolysis (41, 42). Login to comment 170 ABCC7 p.Gly551Asp G551D-CFTR channels in excised macropatches behave much like the èc;1198-CFTR truncation mutant; very low ATP-independent currents under control conditions and strong activation by curcumin (16, 27). Login to comment 171 ABCC7 p.Pro355Ala Additive GOF Effects of the P355A Mutation and a Mutation in the Outer TM Collar-We previously reported another class of GOF mutation that locates to the base of TM9 (Lys-978) in the putative outer TM collar that surrounds the principal pore-lining TMs (see Fig. 1A for predicted location of Lys-978 in CFTR structural models) (16). Login to comment 172 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala Additive GOF Effects of the P355A Mutation and a Mutation in the Outer TM Collar-We previously reported another class of GOF mutation that locates to the base of TM9 (Lys-978) in the putative outer TM collar that surrounds the principal pore-lining TMs (see Fig. 1A for predicted location of Lys-978 in CFTR structural models) (16). Login to comment 173 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala We reasoned that, given the different locations of the two classes of GOF mutations (pore versus outer collar), a double mutant (P355A/K978C) may exhibit an additive GOF effect. Login to comment 174 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala GOF mutations in the pore-lining TM6 (P355A) and in the outer TM collar (K978C) have additive effects on CFTR channel activity. Login to comment 175 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala GOF mutations in the pore-lining TM6 (P355A) and in the outer TM collar (K978C) have additive effects on CFTR channel activity. Login to comment 176 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala A, macroscopic current record showing the relatively large ATP-independent current and robust activation by 2 mM AMP-PNP for the P355A/K978C double mutant.Fortherampprotocol,conditionswerethesameasforFigs.2-4.BandC,meanfractionalATP-freecurrentandrelativeAMP-PNPactivationnormalized to the control current at 1.5 mM ATP for the indicated single and double mutants. Login to comment 179 ABCC7 p.Pro355Ala The P355A substitution increases the channel activities of CFTR mutant constructs that cannot be activated by ATP. Login to comment 180 ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala The P355A substitution increases the channel activities of CFTR mutant constructs that cannot be activated by ATP. Login to comment 181 ABCC7 p.Pro355Ala A and B, macroscopic currents mediated by èc;1198-CFTR without or with the P355A substitution. Login to comment 183 ABCC7 p.Pro355Ala Note the much larger control current for P355A/èc;1198-CFTR that was insensitive to the addition of the ATP scavenger (i.e. was ATP-independent). Login to comment 184 ABCC7 p.Gly551Asp ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala Note the much larger control current for P355A/èc;1198-CFTR that was insensitive to the addition of the ATP scavenger (i.e. was ATP-independent). Login to comment 185 ABCC7 p.Gly551Asp ABCC7 p.Pro355Ala C and E, scatter plots showing the generally larger macroscopic control currents at afa;80 mV for èc;1198-CFTR channels and G551D-CFTR channels containing the P355A substitution. Login to comment 187 ABCC7 p.Pro355Ala The much lower relative activation of the P355A mutants is due to their higher control or baseline currents. Login to comment 188 ABCC7 p.Pro355Ala The much lower relative activation of the P355A mutants is due to their higher control or baseline currents. Login to comment 190 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala G, immunoblot of P355A-èc;1198, P355A-G551D, and P355A/K978C double mutants transiently expressed in HEK-293T cells. Login to comment 191 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala ABCC7 p.Pro355Ala G, immunoblot of P355A-èc;1198, P355A-G551D, and P355A/K978C double mutants transiently expressed in HEK-293T cells. Login to comment 196 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala The double mutant (P355A/K978C-CFTR) exhibited relatively high ATP-independent currents in excised macropatches (b03;35% of the ATP control current) and strong activation by AMP-PNP that approached that by ATP (b03;90% of the ATP control current). Login to comment 197 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala The double mutant (P355A/K978C-CFTR) exhibited relatively high ATP-independent currents in excised macropatches (b03;35% of the ATP control current) and strong activation by AMP-PNP that approached that by ATP (b03;90% of the ATP control current). Login to comment 198 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala It is also evident from the single mutant data in Fig. 5, B and C, that the K978C mutation had a somewhat stronger GOF effect than the P355A mutation. Login to comment 199 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala It is also evident from the single mutant data in Fig. 5, B and C, that the K978C mutation had a somewhat stronger GOF effect than the P355A mutation. Login to comment 200 ABCC7 p.Pro355Ala The P355A TM6 Mutation Increases the ATP Sensitivity of Channel Gating-A GOF or isomerization mutation is predicted by a classical allosteric activation scheme to enhance ligand occupancy at normally subsaturating concentrations. Login to comment 201 ABCC7 p.Pro355Ala The P355A TM6 Mutation Increases the ATP Sensitivity of Channel Gating-A GOF or isomerization mutation is predicted by a classical allosteric activation scheme to enhance ligand occupancy at normally subsaturating concentrations. Login to comment 202 ABCC7 p.Tyr1219Gly ABCC7 p.Pro355Ala The data in Fig. 6 confirm this prediction both for the single P355A mutant (Fig. 6A) and for a double mutant in which the Pro-355 mutation was introduced into an NBD2 mutant that has a markedly reduced ATP affinity (Y1219G-CFTR; Fig. 6B). Login to comment 203 ABCC7 p.Tyr1219Gly ABCC7 p.Pro355Ala The data in Fig. 6 confirm this prediction both for the single P355A mutant (Fig. 6A) and for a double mutant in which the Pro-355 mutation was introduced into an NBD2 mutant that has a markedly reduced ATP affinity (Y1219G-CFTR; Fig. 6B). Login to comment 205 ABCC7 p.Tyr1219Gly As reported previously by the latter authors, the Y1219G mutant of CFTR exhibited a marked rightward shift in the ATP dose-response curve relative to wild type CFTR with an EC50 of 1.5-2 mM. Login to comment 206 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Pro355Ala As reported previously by the latter authors, the Y1219G mutant of CFTR exhibited a marked rightward shift in the ATP dose-response curve relative to wild type CFTR with an EC50 of 1.5-2 mM. Login to comment 207 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala Introducing the P355A mutation increased the apparent ATP affinity of the Y1219G mutant (leftward shift in Fig. 6B). Login to comment 208 ABCC7 p.Tyr1219Gly ABCC7 p.Lys978Cys The K978C mutation in the outer TM collar (TM9) also increased the apparent ATP affinity of the Y1219G mutant and to a greater degree than the Pro-355 substitution, as would be expected if the former is a stronger GOF mutation (see also Fig. 5). Login to comment 209 ABCC7 p.Lys978Cys These results also indicate that both classes of GOF mutations (Pro-355 in TM6; K978C in the outer TM collar) can compensate for a partial defect in ATP binding by allosteric coupling between the TMs and the NBDs (i.e. by "allosteric rescue" of the ATP binding defect). Login to comment 210 ABCC7 p.Tyr1219Gly ABCC7 p.Lys978Cys These results also indicate that both classes of GOF mutations (Pro-355 in TM6; K978C in the outer TM collar) can compensate for a partial defect in ATP binding by allosteric coupling between the TMs and the NBDs (i.e. by "allosteric rescue" of the ATP binding defect). Login to comment 211 ABCC7 p.Tyr1219Gly Homologous TM Mutations Rescue Defective Substrate Export by ATP Binding Mutants of a Yeast MRP-Our finding that an ATP binding mutant of CFTR (Y1219G in NBD2) was rescued by GOF mutations in the TMs motivated us to determine whether such allosteric coupling between the TMs and NBDs could also be observed for an MRP drug exporter. Login to comment 214 ABCC7 p.Pro355Ala A, ATP titration curves for WT and P355A-CFTR channels averaged over five and four macropatch experiments, respectively. Login to comment 215 ABCC7 p.Pro355Ala A, ATP titration curves for WT and P355A-CFTR channels averaged over five and four macropatch experiments, respectively. Login to comment 216 ABCC7 p.Pro355Ala Symbols, means afe; S.E. (error bars); curves, best fits to the Hill equation (WT, K afd; 123.2 òe;M and Hill coefficient afd; 0.83; P355A, K afd; 47.7 òe;M and Hill coefficient afd; 1.01). Login to comment 217 ABCC7 p.Pro355Ala Symbols, means afe; S.E. (error bars); curves, best fits to the Hill equation (WT, K afd; 123.2 òe;M and Hill coefficient afd; 0.83; P355A, K afd; 47.7 òe;M and Hill coefficient afd; 1.01). Login to comment 219 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala Curves, best fits to Hill equation with K values of 1522, 683, and 180 òe;M and Hill coefficients of 1.69, 1.93, and 1.72 for Y1219G (n afd; 6 patches), P355A/Y1219G (n afd; 5), and K978C/Y1219G (n afd; 4), respectively. Login to comment 220 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala Curves, best fits to Hill equation with K values of 1522, 683, and 180 òe;M and Hill coefficients of 1.69, 1.93, and 1.72 for Y1219G (n afd; 6 patches), P355A/Y1219G (n afd; 5), and K978C/Y1219G (n afd; 4), respectively. Login to comment 221 ABCC7 p.Tyr1219Gly *, p b0d; 0.05 compared with Y1219G by unpaired t test. Login to comment 232 ABCC7 p.Tyr1219Gly Yor1p mutations at positions Pro-485 (TM6) and Lys-997 (TM9) that are homologous to the CFTR GOF mutations described above were assayed both as single mutants and as double mutants when combined with one of two NBD mutations that are expected to inhibit Mg-ATP binding to Yor1p: (i) Y1222G, an A-loop mutation homologous to the Y1219G mutation of CFTR, and (ii) D734N, a Walker B mutation that is predicted to reduce Mg-ATP binding because the conserved aspartate helps coordinate the metal cofactor in ABC exporters (13, 46, 47). Login to comment 233 ABCC7 p.Asp572Asn ABCC7 p.Tyr1219Gly Yor1p mutations at positions Pro-485 (TM6) and Lys-997 (TM9) that are homologous to the CFTR GOF mutations described above were assayed both as single mutants and as double mutants when combined with one of two NBD mutations that are expected to inhibit Mg-ATP binding to Yor1p: (i) Y1222G, an A-loop mutation homologous to the Y1219G mutation of CFTR, and (ii) D734N, a Walker B mutation that is predicted to reduce Mg-ATP binding because the conserved aspartate helps coordinate the metal cofactor in ABC exporters (13, 46, 47). Login to comment 234 ABCC7 p.Asp572Asn Unfortunately, the corresponding CFTR Walker B mutant (D572N) is a severe endoplasmic reticulum processing mutant that could not be analyzed by patch clamping in HEK cells (48). Login to comment 251 ABCC7 p.Gly551Asp Each of several mutations at this position exhibited GOF effects on channel gating that were evidenced as follows: (i) higher Po values and more frequent channel openings in the absence of nucleotide; (ii) substantially stronger activation by the normally weak agonist, AMP-PNP; (iii) functional rescue of CFTR channel constructs that normally cannot be activated by ATP (i.e. èc;1198- and G551D-CFTR), and (iv) increased ATP sensitivity of channel gating either alone or when combined with an NBD2 mutation that partially inhibits ATP binding. Login to comment 252 ABCC7 p.Gly551Asp Each of several mutations at this position exhibited GOF effects on channel gating that were evidenced as follows: (i) higher Po values and more frequent channel openings in the absence of nucleotide; (ii) substantially stronger activation by the normally weak agonist, AMP-PNP; (iii) functional rescue of CFTR channel constructs that normally cannot be activated by ATP (i.e. èc;1198- and G551D-CFTR), and (iv) increased ATP sensitivity of channel gating either alone or when combined with an NBD2 mutation that partially inhibits ATP binding. Login to comment 283 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala GOF effects of the P355A and K978C mutations were additive, implying that they influence channel gating by different mechanisms. Login to comment 284 ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala GOF effects of the P355A and K978C mutations were additive, implying that they influence channel gating by different mechanisms. Login to comment 303 ABCC7 p.Tyr1219Gly This is supported by the strong effects of the two classes of GOF mutations on the ATP sensitivity of Y1219G-CFTR activity. Login to comment 304 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly This is supported by the strong effects of the two classes of GOF mutations on the ATP sensitivity of Y1219G-CFTR activity. Login to comment 305 ABCC7 p.Tyr1219Gly The Y1219G mutation is located within a conserved aromatic region known as the A-loop that lies just upstream of the Walker A motif in NBD2. Login to comment 307 ABCC7 p.Tyr1219Gly ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala Both the P355A and K978C GOF mutations increased the ATP sensitivity of the Y1219G mutant with the K978C substitution restoring the ATP sensitivity of channel gating to nearly wild type levels. Login to comment 308 ABCC7 p.Tyr1219Gly ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Pro355Ala Both the P355A and K978C GOF mutations increased the ATP sensitivity of the Y1219G mutant with the K978C substitution restoring the ATP sensitivity of channel gating to nearly wild type levels. Login to comment 314 ABCC7 p.Tyr1219Gly The other ATP binding mutant (Y1222G-Yor1p) was homologous to the A-loop Y1219G mutant in CFTR NBD2 that exhibits markedly reduced ATP sensitivity. Login to comment 315 ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly The other ATP binding mutant (Y1222G-Yor1p) was homologous to the A-loop Y1219G mutant in CFTR NBD2 that exhibits markedly reduced ATP sensitivity. Login to comment 316 ABCC7 p.Tyr1219Gly As discussed above, the ATP sensitivity of Y1219G-CFTR was enhanced by both classes of GOF mutation, an effect we interpreted as allosteric rescue of an ATP binding defect by such TM mutations. Login to comment 323 ABCC7 p.Gly551Asp This thermodynamic distinction probably explains why TM mutations can enhance the activities of G551D-CFTR channels (Fig. 4), whereas the homologous mutations cannot functionally rescue the corresponding Yor1p signature sequence mutant (Fig. 7G). Login to comment 324 ABCC7 p.Gly551Asp This thermodynamic distinction probably explains why TM mutations can enhance the activities of G551D-CFTR channels (Fig. 4), whereas the homologous mutations cannot functionally rescue the corresponding Yor1p signature sequence mutant (Fig. 7G). Login to comment