ABCMdb

PMID: 22680785

Liu X, O'Donnell N, Landstrom A, Skach WR, Dawson DC
Thermal instability of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity.
Biochemistry. 2012 Jun 26;51(25):5113-24. Epub 2012 Jun 15., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K, and R1070W, but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Login to comment 6 ABCC7 p.Lys1250Ala Another mutation, K1250A, known to increase open probability (Po) of ΔF508 CFTR channels, exacerbated thermal inactivation. Login to comment 13 ABCC7 p.Ile539Thr ABCC7 p.Arg555Lys Consistent with this hypothesis, low-temperature rescued ΔF508 CFTR channels exposed to 37 °C exhibit a markedly reduced metabolic half-life (t1/2 < 4 h versus t1/2 > 24 h for wt CFTR14-17,21 ) and rapid thermal inactivation of chloride channel function.5,22 ΔF508 CFTR folding defects can also be suppressed to varying degrees by a variety of second-site mutations in NBD1.4,8,18,23-30 I539T, occurring naturally in many CFTR orthologs, improved the maturation of ΔF508 CFTR at 37 °C,4,25,30 but actually reduced open probability (Po) determined in detached patches.9 Another, R555K, modestly improved protein processing but also increased Po of ΔF508 CFTR channels in detached patches. Login to comment 14 ABCC7 p.Arg553Met In contrast, the suppressor Received: January 6, 2012 Revised: June 6, 2012 Published: June 8, 2012 Article pubs.acs.org/biochemistry (c) 2012 American Chemical Society 5113 dx.doi.org/10.1021/bi300018e | Biochemistry 2012, 51, 5113-5124 mutation R553M was reported to be only modestly effective at correcting either defect.4,23 Thus, while second-site mutations can confer different phenotypes on ΔF508 CFTR with respect to maturation and channel function, the precise mechanisms by which they impact intramolecular interactions within, and external to, NBD1 remain poorly understood. Login to comment 18 ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys We identified unique functional signatures for five second-site mutations, four in NBD1 (I539T, G550E, R553M, and R555K) and one in the fourth intracellular loop (ICL4, R1070W), and also investigated the relation of thermal stability to variations in channel gating brought about by intracellular cAMP, CFTR potentiators, and CFTR inhibitors. Login to comment 19 ABCC7 p.Ile539Thr ABCC7 p.Arg553Met Consistent with previous studies, ΔF508 CFTR-mediated conductance, rescued by incubating oocytes at room temperature, decreased rapidly at 37 °C.5,22 When ΔF508 CFTR was expressed in the context of single, second site mutations, however, results ranged from complete protection from thermal inactivation at 37 °C (R553M) to profound inactivation that was fully reversed upon returning the bath to room temperature (I539T). Login to comment 74 ABCC7 p.Arg334Cys Additional covalent labeling experiments conducted with R334C/ΔF508 CFTR channels indicated that potentiation of the thermally inactivated conductance at 22 °C by P2 was not attributable to the addition of new channels to the membrane and must therefore reflect a P2-induced increase in channel open probability consistent with that reported by Pedemonte et al.36 (see Supporting text and Figure S2). Login to comment 124 ABCC7 p.Arg553Met Of the four NBD1 suppressor mutations tested only one, R553M, fully restored wt thermostability to ΔF508 CFTR channels. Login to comment 125 ABCC7 p.Arg553Met ABCC7 p.Arg555Lys In contrast, pairing ΔF508 with R555K, a mutation that has been reported to be somewhat more effective than R553M at improving NBD1 folding and protein maturation,4,6,24 resulted in a channel that, although unable to sustain the initial increase in conductance evoked at 37 °C, was inactivated only slightly and returned to its prewarming level relatively rapidly when superfusate temperature was returned to 22 °C. Login to comment 126 ABCC7 p.Gly550Glu More pronounced inactivation was seen in G550E/ Table 1. Login to comment 135 ABCC7 p.Arg553Met Representative experiments for (A) R553M/ΔF508 CFTR (n = 3). Login to comment 137 ABCC7 p.Arg555Lys (B) R555K/ΔF508 CFTR (n = 5). Login to comment 139 ABCC7 p.Gly550Glu (C) G550E/ ΔF508 CFTR (n = 4). Login to comment 141 ABCC7 p.Ile539Thr (D) I539T/ΔF508 CFTR (n = 5). Login to comment 144 ABCC7 p.Ile539Thr ΔF508 CFTR and I539T/ΔF508 CFTR, but in both cases the conductance decrease at 37 °C was followed by complete recovery at 22 °C. Login to comment 146 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys There was no apparent correlation of the functional phenotype of the double mutant channels at 37 °C with the improvements reported for NBD1 folding and protein maturation,4,6 but the partial protection from thermal inactivation by R555K and G550E suggested that the effects might be correlated with the induction of increased Po.8,24 R553M, however, had been reported by Teem et al.24 not to increase Po of ΔF508 channels (34-36 °C), so we investigated the behavior of the double mutant in inside-out patches. Login to comment 147 ABCC7 p.Arg553Met We found that R553M/ΔF508 CFTR, like wt, exhibited a stable increase in Po at 35 °C (Figure 6). Login to comment 148 ABCC7 p.Arg553Met Po of R553M/ΔF508 CFTR channels was comparable to that of ΔF508 channels at 22 °C and increased similarly during the first minute of exposure to 35 °C. Login to comment 150 ABCC7 p.Arg1070Trp Protection from thermal inactivation by mutations that increased the open probability of ΔF508 CFTR channels led us to examine the thermal behavior of R1070W/ΔF508 CFTR. Login to comment 151 ABCC7 p.Arg1070Trp The substitution of this aromatic side chain in ICL4 increased the open probability of the double mutant 7 and was proposed to act, at least in part, by repairing defective coupling between NBD1 and ICL4.6,7 Results depicted in Figure 7A show that pairing ΔF508 with R1070W improved the thermal stability of the double mutant, but did not restore wt-like thermal stability. Login to comment 153 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu After two exposures to the elevated temperature, recovery, although evident, was much slower than that seen with G550E/ΔF508 or I539T/ΔF508 CFTR. Login to comment 154 ABCC7 p.Arg1070Trp ABCC7 p.Arg555Lys Pairing R1070W and a second NBD1 suppressor, R555K, with ΔF508, however, resulted in thermal stability that was indistinguishable from that of wt CFTR (Figure 7B). Login to comment 155 ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr In contrast, combining ΔF508 with R1070W and I539T resulted in channels that could not sustain the elevated conductance seen immediately after warming to 37 °C but were nevertheless able to sustain a substantial conductance at 37 °C (Figure 7C). Login to comment 159 ABCC7 p.Arg553Met R553M/ΔF508 CFTR single-channels recorded from an inside-out patch using symmetric solutions (146 mM [Cl]- ) at pH 7.4. Login to comment 165 ABCC7 p.Arg553Met (H) Summary of changes in NPo for R553M/ΔF508 CFTR with time before and during warming to 35 °C. Login to comment 168 ABCC7 p.Arg1070Trp Protection of ΔF508 CFTR from thermal inactivation by R1070W. Login to comment 169 ABCC7 p.Arg1070Trp (A) Following stimulation, an oocyte expressing R1070W/ ΔF508 CFTR (n = 4) was warmed to 37 °C (gray bar and circles) for 10 min twice in succession. After cooling to 22 °C, the oocyte was exposed to 10 μM CF172 (black bar and circles). Login to comment 170 ABCC7 p.Arg1070Trp ABCC7 p.Arg555Lys (B) Following stimulation, an oocyte expressing R555K/R1070W/ΔF508 CFTR (n = 4) was warmed to 37 °C for 10 min twice. After cooling to 22 °C, the oocyte was exposed to 10 μM CF172. Login to comment 171 ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr (C) Following stimulation, an oocyte expressing I539T/R1070W/ΔF508 CFTR (n = 4) was warmed to 37 °C for 10 min twice. After cooling to 22 °C, the oocyte was exposed to 10 μM CF172. Login to comment 179 ABCC7 p.Lys1250Ala K1250A/ΔF508/CFTR Channels Inactivate More Rapidly at 37 °C. Login to comment 180 ABCC7 p.Lys1250Ala The Po of ΔF508 CFTR channels can be enhanced by substitutions for the "Walker lysine" in NBD2 (K1250) which slow the hydrolysis of ATP at composite site 2 of the NBD1/NBD2 dimer47 such that channels exhibit prolonged open times at room temperature.47 The results depicted in Figure 9 show that channel function of the double mutant (K1250A/ΔF508 CFTR) is even less stable than ΔF508 CFTR as judged by the increased rate of thermal inactivation. Login to comment 202 ABCC7 p.Lys1250Ala K1250A accelerated thermal inactivation of ΔF508 CFTR. Login to comment 203 ABCC7 p.Lys1250Ala (A) After stimulation, an oocyte expressing ΔF508 CFTR (gray bar and circles) or K1250A/ΔF508 CFTR (gray bar and downward triangles) was warmed to 37 °C for 10 min and then the bath was cooled to 22 °C. Login to comment 204 ABCC7 p.Lys1250Ala (B) Summary of the half-time of thermal inactivation of K1250A/ΔF508 and ΔF508 CFTR. Login to comment 246 ABCC7 p.Arg553Met From this perspective, therefore, it was somewhat surprising that a single, second-site mutation, R553M, reported to only modestly improve ΔF508 protein maturation at 37 °C,4,6,23 nevertheless fully restored wt-like thermal stability to ΔF508 CFTR channel function. Login to comment 247 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Similarly, G550E, R555K, and R1070W; when combined individually with ΔF508, improved protein maturation at 37 °C to at most 18% of wt,4,6 but nevertheless significantly improved the thermal stability of the double mutant channels. Login to comment 248 ABCC7 p.Ile539Thr In contrast, I539T, which also slightly improved ΔF508 CFTR maturation in mammalian cells, failed to protect ΔF508 CFTR channels from profound thermal inactivation at 37 °C, although the double mutant recovered fully when returned to room temperature. Login to comment 251 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys The three NBD1, second-site mutations that fully or partially protected ΔF508 CFTR channels from thermal inactivation at 37 °C, R553M, R555K, and G550E, share a common effect on ΔF508 CFTR channel function. Login to comment 252 ABCC7 p.Arg553Met They either maintain (R553M, present work) or increase (G550E8 , R555K24 ) the open probability of ΔF508 CFTR channels at 37 °C. Login to comment 254 ABCC7 p.Arg553Met ABCC7 p.Arg555Lys Mendoza et al.6 reported that these three NBD1 suppressor mutations increased the yield of folded ΔF508 NBD1 in a cell-based assay from 0% (R553M) to 60% (R555K), although even a 60% increase represented less than 20% of the yield of wt protein under the same conditions. Login to comment 256 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys A fourth NBD1 suppressor mutation, I539T, in contrast to G550E, R553M, and R555K, is predicted to lie within an unstructured linker connecting two α-helical portions of NBD1. Login to comment 259 ABCC7 p.Ile539Thr ABCC7 p.Gly550Glu ABCC7 p.Arg555Lys Recovery from partial inactivation was also seen with R555K and G550E, but unlike I539T, both of these second-site mutations also resulted in persistent, steady-state conductance at 37 °C. Login to comment 260 ABCC7 p.Ile539Thr The basis for this difference may be apparent in the gating behavior of I539T/ΔF508 channels. Login to comment 261 ABCC7 p.Ile539Thr Recently, Dong et al.9 reported that the open probability of I539T/ΔF508 channels, studied at room temperature in patches detached from HeLa cells, was only about 20% of that of ΔF508 channels, due primarily to prolonged interburst intervals. Login to comment 262 ABCC7 p.Ile539Thr This result indicates that I539T, although it appears to significantly improve the folding of ΔF508 NBD1 in a cell-based assay,6 actually further reduces open probability of the double mutant channel, even at room temperature. Login to comment 263 ABCC7 p.Ile539Thr This observation is consistent with the previous report of DeCarvalho et al.25 that I539T/ΔF508, although it exhibited somewhat improved protein processing, was actually inferior to ΔF508 CFTR in a forskolin dose-response assay. Login to comment 265 ABCC7 p.Arg1070Trp The ICL4 mutation, R1070W, increased the thermal stability of ΔF508 CFTR channel function, although it did not fully restore the wt-like behavior at 37 °C. Login to comment 266 ABCC7 p.Arg1070Trp ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys Like G550E, R553M, and R555K, this second-site mutation has been associated with increased open probability of the double mutant,7 an effect attributed to a partial improvement in the interaction between NBD1 and ICL4.29,57 Combining the ICL4 mutation with an NBD1 suppressor mutation on the ΔF508 background (R555K/R1070W/ΔF508), however, fully restored wt-like thermal stability at 37 °C, an "additive" effect similar to that reported by Mendoza et al 6 in their study of the effect of these mutations on NBD1 folding and ΔF508 CFTR protein yield. Login to comment 267 ABCC7 p.Arg1070Trp ABCC7 p.Arg553Met The rescue of wt thermal stability of channel function by the R553M mutation, however, indicates that the structural modifications introduced by combining ΔF508 with R1070W are not required for the thermal stabilization of channel gating. Login to comment 268 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr Mendoza et al.6 also reported that combining R1070W with I539T, which alone increased the cellular yield of NBD1 to about 80% of wt, resulted in a yield of the I539T/R1070W/ ΔF508 protein that was 76% of the wt level. Login to comment 269 ABCC7 p.Ile539Thr We found that these channels, although apparently more thermostable that ΔF508 CFTR, nevertheless failed to exhibit full, wt-like thermostability of channel function, a result that is consistent with the adverse effect of the I539T second-site mutation on open probability.9 Channel Gating and Functional Stability of ΔF508 CFTR. Login to comment 274 ABCC7 p.Lys1250Ala Likewise, two experimental maneuvers that increased channel open probability, a second site mutation in NBD2 (K1250A) and a CFTR potentiator (P2), actually exacerbated thermal inactivation, although two other potentiators (P1 and Genistein) did not. Login to comment 277 ABCC7 p.Ile539Thr In addition, the slowing of the time course of stimulation of ΔF508 CFTR channels (lacking any second-site mutation) following a pre-stimulation thermal challenge is consistent with a model in which, at 37 °C, unstimulated ΔF508 CFTR channels that are rarely if ever transitioning to an open state, can nevertheless be driven into an inactivated state, but like I539T/ΔF508 CFTR, one from which they can recover spontaneously under stimulating conditions at 22 °C. Login to comment 285 ABCC7 p.Arg334Cys ■ ASSOCIATED CONTENT *S Supporting Information Supporting text and Figures S1 and S2 document the lack of major insertion of new, unlabeled R334C/ΔF508 CFTR channels to the membrane during cAMP stimulation, recovery following warming (Figure S1) and potentiation by PG-01 (P2) following warming (Figure S2). Login to comment