ABCMdb

PMID: 21965669

Wang W, Okeyo GO, Tao B, Hong JS, Kirk KL
Thermally unstable gating of the most common cystic fibrosis mutant channel (DeltaF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops.
J Biol Chem. 2011 Dec 9;286(49):41937-48. Epub 2011 Sep 30., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys Here, we determined whether these various rescue protocols induce a ⌬F508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ⌬F508-CFTR function. Login to comment 46 ABCC7 p.Arg555Lys ABCC7 p.Arg29Lys For example, Hegedus et al. have shown that eliminating two arginine-based motifs (RXR) from ⌬F508-CFTR (e.g. R29K and R555K) promotes maturation of ⌬F508, but channel activity in lipid bilayers is highly thermally unstable (i.e. inactivates at physiologic temperature) (42). Login to comment 52 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys In a recent study we showed that two cytosolic loop mutations (e.g. K190C and K978C) promote ATP-independent CFTR channel activity, allosterically increase ATP and PKA sensitivity, and also significantly restore the function of mutant CFTR channels that cannot be activated by ATP binding or NBD dimerization (e.g. G551D and ⌬1198, which lacks NBD2) (47). Login to comment 57 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys The cytosolic loop mutations (K978C and K190C/K978C) do not improve ⌬F508-CFTR processing but do increase the ATP-independent channel activity of ⌬F508 channels that are delivered to the cell surface by incubation at low temperature. Login to comment 64 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys K978C and K190C/K978C mutations, which promote ATP-independent channel activity (47), were introduced into the ⌬F508-CFTR construct. Login to comment 65 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys The ⌬F508-CFTR construct with NBD1 suppressor mutations (G550E, R553M, R555K (3M/⌬F508)) was provided by Dr. Phillip Thomas (University of Texas Southwestern Medical Center, Dallas). Login to comment 137 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys Suppressor Mutations in NBD1 Correct Misfolding and Stabilize ⌬F508-CFTR Channel Activity at 36.5 °C-We have shown recently that three suppressor mutations (G550E, R553M, R555K (3M/⌬F508)) in NBD1 correct ⌬F508-CFTR maturation and misfolding and markedly increase its channel activity in excised patches at room temperature (43). Login to comment 162 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys Constitutive Loop Mutations Increase ATP-independent Channel Activity of ⌬F508-CFTR, Which Is More Stable at Physiologic Temperature-Previously, we showed that several mutations in intracellular loop1 and loop3 (e.g. K190C and K978C) increase the ATP-independent channel activities of several CFTR constructs, including mutant forms that cannot be activated by ATP (e.g. G551D and ⌬1198-CFTR, an NBD2 deletion mutant) (47). Login to comment 164 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys Fig. 6A shows that neither K978C nor K190C/K978C improved the maturation of ⌬F508-CFTR when cells were grown either at 37 °C or 27 °C, as determined by immunoblotting. Login to comment 169 ABCC7 p.Lys978Cys Fig. 7A shows that K978C/⌬F508-CFTR exhibited a partial decrease of current when the bath temperature was elevated to 36.5 °C, but on average 30-40% of this current remained after warming the bath (see Fig. 7D). Login to comment 170 ABCC7 p.Lys978Cys Interestingly, for approximately 50% of the patches the current slightly recovered when the temperature returned to room temperature, indicating that some of the current decrease for K978C/⌬F508-CFTR was reversible. Login to comment 171 ABCC7 p.Lys978Cys These results indicate that K978C partially protects ⌬F508 channel activity from thermal inactivation. Login to comment 172 ABCC7 p.Lys978Cys We then added hexokinase/glucose to remove ATP to determine whether the remaining current is ATP-independent. As shown in Fig. 7A, most of the current that remained following warming the bath was insensitive to removal of ATP, indicating that the ATP-independent channel activity of K978C/⌬F508-CFTR is far more stable at 36.5 °C compared with that of ⌬F508-CFTR alone (Fig. 1B). Login to comment 173 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys To follow up this result we examined the thermal stability of the K190C/K978C/⌬F508 construct that has a greater relative ATP-independent channel activity. Login to comment 174 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys Fig. 7B shows that the K190C/K978C double mutation greatly protected the channel activity from thermal inactivation with nearly all of the remaining current following warming the bath being ATP-independent. As a control we also tested the thermal stability of the ATP-independent channel activity of G551D-CFTR channels with the K978C mutation. Login to comment 175 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys Fig. 7C shows that raising the temperature did not inactivate the K978C/G551D-CFTR current, indicating that the ATP-independent channel activity of K978C/G551D is thermally stable. Login to comment 180 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys Cells expressing G550E/R553M/R555K/⌬F508 (3M/⌬F508) were grown at 37 °C. Login to comment 185 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys Error bars, S.E. Thermal Instability of ⌬F508-CFTR Gating DECEMBER 9, 2011•VOLUME 286•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 41943 the ATP-independent channel activity of K978C/⌬F508-CFTR and to a greater extent K190C/K978C/⌬F508-CFTR was resistant to temperature-induced inactivation. Login to comment 188 ABCC7 p.Lys978Cys To examine this idea further we recorded the unitary currents mediated by single K978C/⌬F508-CFTR channels in micropatches obtained with small tip pipettes. Login to comment 190 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys Fig. 8E shows that the amplitudes of the singlechannelcurrentsforK978C/⌬F508afterincubationat36 °C were similar to those for WT- and K978C-CFTR (47), indicating that the conformation of the pore for K978C/⌬F508 is not obviously compromised at physiologic temperature. Login to comment 195 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys We found that constitutive mutations in cytosolic loop1 and loop3 (i.e. K978C and K190C/K978C) increased ATP-independent channel activity of low temperature-rescued ⌬F508-CFTR. Login to comment 199 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys FIGURE6.Cytosolicloopmutations(K978CandK190C/K978C)increaseATP-independentchannelactivityoflowtemperature-recued⌬F508-CFTR.A, immunoblot showing that loop mutations do not improve maturation of ⌬F508 at 27 °C. B-D, ATP-dependent channel activities of ⌬F508, K978C/⌬F508, and K190C/K978C/⌬F508 revealed by adding hexokinase/glucose to scavenge ATP. Login to comment 201 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys E, mean fractional ATP-independent currents for ⌬F508 (n ϭ 6), K978C/⌬F508 (n ϭ 5), and K190C/K978C/⌬F508-CFTR (n ϭ 5). Login to comment 218 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys K978C/⌬F508 and K190C/K978C/⌬F508-CFTR are more stable at physiologic temperature. Login to comment 219 ABCC7 p.Lys978Cys A, effect of raising temperature on K978C/⌬F508. Login to comment 221 ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys B, effect of raising temperature on K190C/K978C/⌬F508-CFTR, which is more thermally stable. Login to comment 222 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys C, effect of raising temperature on K978C/G551D-CFTR. Login to comment 223 ABCC7 p.Gly551Asp ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys D, mean fractional current decrease for ⌬F508 (data from Fig. 1D), K978C/⌬F508, K190C/K978C/⌬F508, and K978C/ G551D. Login to comment 237 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys A and B, effect of raising temperature on ATP-independent currents of K978C/⌬F508- and K190C/K978C/⌬F508-CFTR. Login to comment 239 ABCC7 p.Lys978Cys ABCC7 p.Lys978Cys ABCC7 p.Lys190Cys C, mean fractional loss of ATP-independent current for K978C/⌬F508 and K190C/ K978C/⌬F508-CFTR (n ϭ 4 for each construct). Login to comment 240 ABCC7 p.Lys978Cys D, effect of raising temperature on unitary currents of K978C/⌬F508. Login to comment 242 ABCC7 p.Lys978Cys Effect of raising bath temperature on K978C/⌬F508 current was monitored at the macroscopic level (ramp protocol), and then the unitary currents were recorded in the gap-free mode after reduction to room temperature. Login to comment 254 ABCC7 p.Gly551Asp Earlier we showed that these constitutive loop mutations markedly increased channel opening rates for CFTR constructs that cannot respond to ATP (G551D-CFTR) or dimerize the two NBDs (i.e. an NBD2 deletion construct, ⌬1198-CFTR). Login to comment