Cholesterol Interaction Directly Enhances Intrinsic Activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
, Jean-Philippe Julien 1,2,4 and Christine E. Bear 1,2,3,*
Oscar Moran
Round 1
Reviewer 1 Report
Stephanie Chin and coworkers compare the functional properties, ATPase activity and ion transport, of CFTR purified with detergents and surfactants. The conclude that detergents someway damage the CFTR, perhaps because it removes the associated lipids. The role of cholesterol in the functionality of the protein is highlighted.
Perhaps one of the most important aspects of this work is in questioning the goodness of molecular models that have been built with cryo-electron microscopy. The work is very well done, it is consistent and very clear to the reading. It would be convenient to clarify only two points:
1-When the CFTR is reconstituted in POPC vesicles probably a part of the cholesterol and the other phospholipids associated with the protein could be "diluted" in the lipid matrix of the bilayer. Is there any evidence that this does not happen? What happens if cholesterol is added in the vesicles? In other words, how fixed are the lipids to the protein?
2- In the discussion it is said that there is a contradiction due to the fact that the lipids increase is the ATPasic activity that the anionic transport. In my opinion, these two facts are not contradictory; both, ATPasic activity and transport depend on the ATP binding to the protein. It would be important to clarify this point.
Minor corrections
line 138. The number of the figure is wrong
Figure 4A. The legend of the ordinate should not be efflux of iodide, but concentration of iodide. The efflux is the time-derivative of the concentration.
p { margin-bottom: 0.25cm; line-height: 115%; }Author Response
Please see attachment of Letter to the Editor plus response to both reviewers in aggregate. Specific response to Reviewer 1 is directly inserted here.
Major corrections:
1. When the CFTR is reconstituted in POPC vesicles probably a part of the cholesterol and the other phospholipids associated with the protein could be "diluted" in the lipid matrix of the bilayer. Is there any evidence that this does not happen? What happens if cholesterol is added in the vesicles? In other words, how fixed are the lipids to the protein?
We did not directly assess the affinity of CFTR for cholesterol relative to other phospholipids in this study and therefore, cannot rule out the possibility that lipid exchange occurred during the reconstitution into the POPC liposomes. We changed lines 212 to 214 in the revised manuscript to reflect this caveat. “However, the stoichiometry of cholesterol molecules that interact with CFTR following reconstitution into phospholipid liposomes remains unknown.”
2. In the discussion it is said that there is a contradiction due to the fact that the lipids increase is the ATPasic activity that the anionic transport. In my opinion, these two facts are not contradictory; both, ATPasic activity and transport depend on the ATP binding to the protein. It would be important to clarify this point.
As suggested by our reviewer, we revised our statement on Line 265 to read: This “open state” is favoured by ATP binding and disruption of ATP turnover by mutations of the dominant catalytic base, E1371 8,10,11,51. Therefore, our observation that lipids, including cholesterol, increase both ATPase activity and channel opening, may be explained by an increase in ATP binding to the catalytic site in CFTR. Please see revisions to lines 264-267.
Minor corrections:
line 138. The number of the figure is wrong
The number of the figure has been changed to “Figure 3a” at line 134.
Figure 4A. The legend of the ordinate should not be efflux of iodide, but concentration of iodide. The efflux is the time-derivative of the concentration.
p { margin-bottom: 6.25px; line-height: 115%; }
The y-axis of Figure 4A and the figure legend at line 160 has been changed to “Concentration of iodide (µM) effluxed from proteoliposomes normalized for CFTR concentration”. The figure legend has also been corrected, line 160.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Cholesterol interaction directly enhances intrinsic activity of the cystic fibrosis transmembrane conductance regulator (CFTR)
Stephanie Chin, Mohabir Ramjeesingh, Maurita Hung, June Ereño-Oreba, Hong Cui, Onofrio Laselva, Jean-Philippe Julien, Christine E. Bear
This paper describes biochemical studies on purified CFTR preparations. The authors notice that purification procedures including amphipol lipids, rather than detergent, result in a higher ATPase activity and higher anion efflux after reconstitution in proteoliposomes. They conclude that phospholipids and cholesterol might modulate the activity of CFTR. The results might be relevant to the recent cryo-EM structures of full length CFTR. In particular, the structure of the phosphorylated, ATP-bound CFTR, carrying the “constitutively open” mutation E1371Q, does not show an open permeation pathway. The authors suggest this might be related to the detergent solubilization protocol used for protein preparation in the structural studies.
The observations are interesting, and the experiments are carefully conducted. I have some relatively minor suggestions for improvement.
A) Authors could better clarify experimental conditions
For the experiments shown in figure 4, were two different CFTR preparations used (CFTR purified using amphipol vs. CFTR purified using LMNG) or was a single preparation used, then incubated for 1 hour either with amphipol or with LMNG? Different parts of the manuscript convey different impressions. Compare:
- Line 164 "Iodide efflux traces of proteoliposomes reconstituted with P-CFTR pre-treated with Mg-ATP in amphipol and of proteoliposomes reconstituted with P-CFTR pre-treated with Mg-ATP in LMNG ..."
vs
- Line 249 "we directly compared detergent (LMNG) purified CFTR versus amphipol purified CFTR and we showed that the magnitude of CFTR mediated anion conduction, normalized for CFTR protein mass, was approximately two-fold greater for the amphipol preparation."
A clearer definition of the exact metric used in figure 4B would also help. Is “change in efflux rate” the difference between the rates before and after valinomycin addition, or the ratio between the two rates? If the former is true, what are the units used?
B) Authors could indicate more clearly what evidence supports their interpretation, and what does not
When discussing the main conclusion of this study, namely that the increase in anion efflux (per ng of CFTR protein) seen with amphipol preparations compared to LMNG preparations, likely reflects a direct modulating activity of the lipids on CFTR function, the authors mention an “acute effect”:
Line 253 “This difference [2-fold increase in anion efflux] may reflect either an increase in the number of functionally reconstituted CFTR molecules, or an acute increase in the intrinsic activity of each CFTR molecule if its annulus of associated lipids is preserved. Given the acute effect of cholesterol addition to the ATPase activity of CFTR after purification in detergent micelles, we suggest that cholesterol and phospholipids that are co-purified with CFTR by amphipols can directly modulate the intrinsic channel activity of CFTR molecules in each CFTR: amphipol: cholesterol containing proteoliposome.”
If I understand correctly, the “acute effect” is a change seen after a 1-hour, room-temperature incubation in the presence of cholesterol and phospholipids, as described in Fig. 3B and paragraph 4.8. I am not sure this can really be considered “acute”, as for instance, when channel activity is seen to increase within seconds of ivacaftor addition in electrophysiological experiments, clearly suggesting the drug binds to CFTR directly and immediately changes its gating kinetics. Thus, the conclusion that cholesterol and lipids directly enhance channel/ATPase activity rather than affect the fraction of CFTR molecules acquiring a functional conformation, is not very strong. The latter interpretation is not implausible, especially given the heterogeneity of the preparation (see Fig. 1B and lines 92-94). I think the paper would be improved if a clear mention was made, at this point of the discussion, of the fact that only a minority of the CFTR molecules in the preparation is catalytically active, as demonstrated by the displacement of the peak of ATPase activity vs. CFTR protein abundance.
- Line 233 "revealed an important role for cholesterol "
- Line 236 "We showed that addition of cholesterol to detergent purified CFTR enhanced its ATPase function"
Addition of cholesterol alone was not actually tested. If I understand correctly, a significant difference was noted comparing P-CFTR in LMNG detergent vs. P-CFTR in LMNG detergent pretreated with a mix of lipids including cholesterol. Pretreatment with the same mix of lipids without cholesterol did not result in a significant difference from control. To improve accuracy the statements above should be supported by a P-value obtained comparing pretreatment without and with cholesterol.
C) Authors could better integrate their work with current literature on CFTR structure/function.
- Line 219 "To date, its binding site is unknown..." This sentence needs updating: a VX-770-bound CFTR cryo-EM structure has been published (Liu et al, 2019 doi:10.1126/science.aaw7611)
- Line 264: "…our observation that lipids increase both ATPase activity and anion conductance seems to contradict this consensus view of the gating mechanism. "
I do not think there is any contradiction here. For WT-CFTR, it is very clear that channels spend most of the gating cycle time in the long, interburst closures, suggesting that the opening transition is rate-limiting step of the gating cycle. In the consensus model of ATPase-driven CFTR gating, gating and hydrolytic cycles are tightly coupled and the hydrolytic step coincides with termination of a burst. Thus, if lipids were to increase the rate of the opening transition (without affecting the closing rate), both channel open probability (anion efflux) and ATPase Vmax would be increased.
The specific impairment of catalytic activity seen in E1371 mutants is different from that seen in other catalytic site mutants: not only they have reduced ATPase activity, but they also form very stable NBD1/NBD2 dimers. In these mutants, occupancy of the open state is increased because exit from the open state is very slow (slow hydrolysis), but entry into the open state is fast. Different CFTR active site mutants show different changes in ATPase and Po. For instance, for K1250A mutants, in which opening rate is slowed much more than closing rate, Po (and therefore overall anion conductance) is reduced, and ATPase activity is virtually abolished (see e.g. Ramjeesingh et al., 1999).
Minor:
1. Line 29 “CFTR channel activity and anion conduction through its membrane pore is regulated by protein kinase A (PKA) phosphorylation of its regulatory (R) domain as well as ATP binding and hydrolysis…” There is no evidence that anion conduction (single channel conductance) is regulated by PKA phosphorylation and ATP interactions at the NBDs.
2. Line 137 “Vmax of 23.90 ± 1.91 nmol phosphate/mg protein/min versus Vmax of 5.54 ± 0.66 nmol phosphate/mg protein/min respectively, Figure 4a)”. Figure referred to is Figure 3a.
3. Line 228 “Together with the ATPase activity assays, these findings to suggest that the cholesterol extracted with CFTR by amphipols facilitates VX-770 binding…”
4. Line 288 “Interestingly, in this cryo-EM model, 40% of the ivacaftor molecule interacts with the CFTR protein at the elbow-like joint via hydrogen bond interaction with residue: Y304 and interaction with aromatic residues: F312 and F931“. In the cryo-EM structure I think Y304 forms a hydrogen bond with residues in transmembrane helix 8, not with ivacaftor itself. S308 forms a hydrogen bond with ivacaftor.
5. Line 315 "However, the LMNG preparations were used to generate structural models, supporting the relevance of the current studies to understanding structure: function relationships." I am not sure the fact that structure was determined with a similar preparation supports the claim that this study has relevance for structure/function studies.
6. Line 319 “This haS been reported for voltage-gated potassium and sodium channels”
7. Line 352 “Any heavy particulate matter that came down WAS discarded before spinning the membrane suspension…”
Author Response
Response to Reviewer #2:
Please see attached pdf with the letter to the Editor and the responses to both reviewers in aggregate.
Response to Reviewer 2 inserted directly here.
Our reviewer makes an important point regarding the sentence starting at Line 253:
“I think the paper would be improved if a clear mention was made, at this point of the discussion, of the fact that only a minority of the CFTR molecules in the preparation is catalytically active, as demonstrated by the displacement of the peak of ATPase activity vs. CFTR protein abundance… “and it is not implausible the rather than an acute effect, cholesterol is affect the fraction of CFTR molecules acquiring a functional conformation”
As suggested by our reviewer, we revised the manuscript at this point of the paper to mention that only a fraction of the total, amphipol-purified is catalytically active. Please see the revised text starting at lines 252-255.
Our reviewer commented on Line 236 "We showed that addition of cholesterol to detergent purified CFTR enhanced its ATPase function" The reviewer suggests that the accuracy of the statemtn should be improved because addition of cholesterol alone was not actually tested.
Therefore, we revised this statement to read: “We showed that while phospholipids alone had no effect, the addition of cholesterol, together with phospholipids, to detergent purified CFTR enhanced its ATPase function.” Please see the change in line 235.
219 "To date, its binding site is unknown..." This sentence needs updating: a VX-770-bound CFTR cryo-EM structure has been published (Liu et al, 2019 doi:10.1126/science.aaw7611)
We changed this sentence accordingly (line 220-223)
Our reviewer commented on Line 264: "…our observation that lipids increase both ATPase activity and anion conductance seems to contradict this consensus view of the gating mechanism. " I do not think there is any contradiction here. For WT-CFTR, it is very clear that channels spend most of the gating cycle time in the long, interburst closures, suggesting that the opening transition is rate-limiting step of the gating cycle. In the consensus model of ATPase-driven CFTR gating, gating and hydrolytic cycles are tightly coupled and the hydrolytic step coincides with termination of a burst. Thus, if lipids were to increase the rate of the opening transition (without affecting the closing rate), both channel open probability (anion efflux) and ATPase Vmax would be increased.
As suggested by our reviewer, we revised our statement on Line 265 to read: This “open state” is favoured by ATP binding and disruption of ATP turnover by mutations of the dominant catalytic base, E1371 8,10,11,51. Therefore, our observation that lipids, including cholesterol, increase both ATPase activity and channel opening, may be explained by an increase in ATP binding to the catalytic site in CFTR. Please see revisions to lines 264-267.
Minor corrections:
Line 29 “CFTR channel activity and anion conduction through its membrane pore is regulated by protein kinase A (PKA) phosphorylation of its regulatory (R) domain as well as ATP binding and hydrolysis…”. There is no evidence that anion conduction (single channel conductance) is regulated by PKA phosphorylation and ATP interactions at the NBDs.
We changed this statement in Line 29 to remove the phrase “anion conduction”.
Line 137 “Vmax of 23.90 ± 1.91 nmol phosphate/mg protein/min versus Vmax of 5.54 ± 0.66 nmol phosphate/mg protein/min respectively, Figure 4a)”. Figure referred to is Figure 3a.
Figure 4a from line 134 has been changed to Figure 3a.
Line 288 “Interestingly, in this cryo-EM model, 40% of the ivacaftor molecule interacts with the CFTR protein at the elbow-like joint via hydrogen bond interaction with residue:Y304 and interaction with aromatic residues: F312 and F931“. In the cryo-EM structure I think Y304 forms a hydrogen bond with residues in transmembrane helix 8, not with ivacaftor itself S308 forms a hydrogen bond with ivacaftor.
We corrected this statement in the revised text. Please see line 289.
Line 315 "However, the LMNG preparations were used to generate structural models, supporting the relevance of the current studies to understanding structure: function relationships."
We agree with the reviewer that this a weak statement and it has been removed in the revision.
Line 319 “This haS been reported for voltage-gated potassium and sodium channels” This has been changed.
Line 352 “Any heavy particulate matter that came down WAS discarded before spinning the membrane suspension…” This has been changed.
Author Response File: Author Response.pdf
