ABCMdb

ABCC7 p.Pro355Ala

CF databases:	c.1063C>T , p.Pro355Ser (CFTR1) ? , The mutation was detected by DGGE analysis and characterised by direct sequencing. We have seen it only once, in over 2500 control chromosomes from Italian population.
Predicted by SNAP2:	A: D (63%), C: D (63%), D: D (75%), E: D (75%), F: D (80%), G: D (71%), H: D (63%), I: D (75%), K: D (85%), L: D (80%), M: D (71%), N: D (71%), Q: D (59%), R: D (75%), S: D (63%), T: D (71%), V: D (75%), W: D (80%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] Conserved allosteric hot spots in the transmembran... J Biol Chem. 2014 Jul 18;289(29):19942-57. doi: 10.1074/jbc.M114.562116. Epub 2014 May 29. 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., [PMID:24876383]

Abstract [show]
ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

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Sentences [show]
No. Sentence Comment
136 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
144 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 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
148 Fig. 4, B-D, shows that introducing the P355A mutation into the èc;1198-CFTR background created channels with FIGURE 3. Login to comment
158 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
160 C, macroscopic current record showing substantial activation of P355A-CFTR by 2 mM AMP-PNP when added following ATP removal. Login to comment
165 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 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
168 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
172 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 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
175 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 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
180 The P355A substitution increases the channel activities of CFTR mutant constructs that cannot be activated by ATP. Login to comment
181 A and B, macroscopic currents mediated by èc;1198-CFTR without or with the P355A substitution. Login to comment
184 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 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
188 The much lower relative activation of the P355A mutants is due to their higher control or baseline currents. Login to comment
191 G, immunoblot of P355A-èc;1198, P355A-G551D, and P355A/K978C double mutants transiently expressed in HEK-293T cells. Login to comment
197 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
199 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
201 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
203 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
206 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 Introducing the P355A mutation increased the apparent ATP affinity of the Y1219G mutant (leftward shift in Fig. 6B). Login to comment
215 A, ATP titration curves for WT and P355A-CFTR channels averaged over five and four macropatch experiments, respectively. Login to comment
217 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
220 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
284 GOF effects of the P355A and K978C mutations were additive, implying that they influence channel gating by different mechanisms. Login to comment
308 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
135 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 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
147 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 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 C, macroscopic current record showing substantial activation of P355A-CFTR by 2 mM AMP-PNP when added following ATP removal. Login to comment
164 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
167 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
171 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
174 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
179 The P355A substitution increases the channel activities of CFTR mutant constructs that cannot be activated by ATP. Login to comment
183 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
187 The much lower relative activation of the P355A mutants is due to their higher control or baseline currents. Login to comment
190 G, immunoblot of P355A-èc;1198, P355A-G551D, and P355A/K978C double mutants transiently expressed in HEK-293T cells. Login to comment
196 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 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 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 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
214 A, ATP titration curves for WT and P355A-CFTR channels averaged over five and four macropatch experiments, respectively. Login to comment
216 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 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
283 GOF effects of the P355A and K978C mutations were additive, implying that they influence channel gating by different mechanisms. Login to comment
307 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