Structure and Cl^- Conductance Properties of the Open State of Human CFTR

Reviewer #1 (Public review):

Summary:

The goal of this study was to overcome the apparent difficulty in constructing structural models of the open state of the CFTR chloride channel. While several CFTR structural models at near-atomic resolution have been published under a variety of conditions, none of them have demonstrated a pore open across the full dimension of the plasma membrane. Instead, these have routinely been referred to as "near-open" models. In the present study, the authors extended their findings from a prior paper from their group that investigated a series of brief MD simulations, a small number of which exhibited permeation events where chloride ions permeated the pore. This study included massively repeated simulations initiated from these aforementioned Cl permeable conformations. Extensive analysis of the data identified a novel penta-helical structure that comprises the channel pore. This comprehensive study attempted to explain several features of conducting CFTR channels, including single-channel conductance, selectivity, and the mechanisms linking the ATP-induced dimerization of the cytosolic nucleotide-binding domains (NBDs) to the opening of the channel pore (a.k.a., "pore-gating".

Strengths:

The major strength of this study is its comprehensive nature. The approaches applied are cutting-edge and beyond, and are used to explain many different aspects of channel function in CFTR. The strength of evidence is very strong. The paper is extremely well-written, and the arguments are well-supported.

Weaknesses:

The major weakness is that none of the novel conclusions (i.e., those arising solely from this study and not previously published (have been supported by experimental confirmation. That is typical of computational studies such as this.

Reviewer #2 (Public review):

Although recent cryo-EM structures of the CFTR ion channel were reported in a putative open state (ATP-bound, NBD-dimerized), it remains unclear whether these structures explain the conductive properties of the open channel observed in functional experiments. To investigate this, the authors conducted extensive molecular dynamics simulations at different voltages. The simulations are started from snapshots of their prior work, based on the experimental putative open state and including conditions with high negative voltage. Their analysis reveals that the cryo-EM structure represents a near-open metastable state, with most trajectories transitioning to either more closed or more open conformations, leading to the identification of a potential new open state. Permeation rate analysis shows that, unlike the other states, the proposed open state exhibits functional conductive properties of the open channel, although a strong inward rectification, inconsistent with experimental data, is also noted. Further structural analysis and simulations of ATP-unbound closed states offer additional mechanistic insights.

Overall, this work tackles key questions about CFTR: What is the true open conductive state? Does the ATP-bound cryo-EM structure reflect an actual open state? What is the ion permeation mechanism, and what structural changes occur during the closed-to-open transition? Which residues are critical, particularly those linked to diseases like CF? The study, based on a comprehensive set of all-atom molecular dynamics simulations, including a range of physiologically relevant voltages, provides important insights in this regard. It identifies key structural states, permeation pathways, critical residues, and conductance properties that can be directly compared to functional data. Notably, the analysis identifies a new open state of the channel, which, systematic analysis convincingly demonstrates is a conductive conformation of the channel, in line with experimental data at negative voltages. The authors carefully address some of the limitations of their results, exploring and discussing discrepancies with functional experiments, such as inward rectification. The work is also very well written, with a clear and logical presentation of key findings.

The main weakness of this study is that the simulation data rely on the conventional CHARMM36 force field for Cl− ions, which has been shown to significantly underestimate the interaction between Cl− and proteins (J. Chem. Theory Comput. 2021, 17, 6240-6261). For example, the conventional CHARMM36 force field destabilizes the Cl-binding site in CLC-ec1. The latter ion unbinds irreversibly during microseconds-long simulations which is at odds with the experimental binding affinity.

This imbalance in Cl−/protein/water interactions could significantly impact the CFTR simulations, potentially altering state populations and Cl− permeability. Notably, recent work by Levring and Chen (Proc Natl Acad Sci U S A. 2024) identifies a likely Cl− binding site in the bottleneck region of the channel, which contradicts the simulation results showing low occupancy Cl− ions in this region (Fig. 1B and Fig. 6A). This discrepancy may be due to the underestimation of Cl−/protein interactions. Indeed, Orabi et al. have proposed corrections that specifically tune these interactions, including those with aromatic residues, in line with the binding site geometry suggested by Levring and Chen. This imbalance in interactions may also lead to an underestimation of the conductance in the experimental near-open state.
Balanced Cl−/protein interactions could also influence voltage/current relationships, potentially affecting the degree of inward rectification. For example, higher Cl− occupancy in the bottleneck region may stabilize the down state of R334, along with other measured interactions, thereby increasing conductance as the authors have shown.

The experimental evidence reported and discussed by the authors in support of the proposed open state is largely qualitative. For instance, in Figure 4 Supplement 2 there is a significant overlap in the distances and SASA distributions of open and near-open states for the reported residues (are those residues water accessible in the simulations?).

Given the known limitations of the standard CHARMM36 Cl− force field and in the absence of robust experimental validation of the proposed open state, I recommend validating at least part of the results using an independent set of simulations (not started from the previous ones) with an updated Cl− force field. It would be especially important to reassess whether the experimental near-open state is truly metastable and less probable than the new open state, and confirm that the near-open state exhibits negligible conductance.

A minor point worth discussing is whether the observed inward rectification may be influenced by hysteresis or incomplete equilibration, as many simulations were started from prior trajectories at large negative voltages and may not have fully relaxed. For instance, is not uncommon that small structural changes in backbone and sidechains occur in several microseconds (Shaw et al., Science, 2010). That said, discrepancies in current-voltage relationships are not unexpected due to challenges in simulation sampling and force field accuracy (J Gen Physiol 2013 May;141(5):619-32) as the authors stated.

Another minor point to address is the preparation of the simulation setup for the ATP-free structure of the protein. It would be helpful to specify whether any particular controls or steps were taken, given that the structure is based on a relatively low resolution (3.87 Å) model.

Reviewer #3 (Public review):

Background:

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel whose dysfunction underlies cystic fibrosis, a life-limiting condition caused by thick, sticky mucus buildup in the lungs and other organs. Despite multiple high-resolution structures of CFTR, these snapshots have all captured the channel in a non-conducting or "closed" conformation - even when the protein was prepared under conditions that should favor channel opening. This discrepancy has posed a key challenge: how can a channel be experimentally observed as closed while physiological tests demonstrate it conducts chloride ions?

Key Findings:

(1) Stable Open Conformation

Through repeated molecular dynamics (MD) simulations of human CFTR in lipid bilayers, researchers observed a reproducible, stable open state. Unlike previous transient openings seen in single-run or short simulations, this conformation remains consistently permeable over extended timescales.

(2) Penta-Helical Arrangement

The authors highlight a "penta-helical" pore-lining arrangement in which five transmembrane helices symmetrically organize to create a clear ion-conduction pathway. This novel configuration resolves the previously puzzling hydrophobic bottleneck found in cryo-EM structures.

(3) Conductance Close to Experimental Values

By analyzing chloride ion flow under near-physiological voltages, they calculate a channel conductance aligning well with electrophysiological measurements. This alignment provides strong support that the observed structure is functionally relevant.

(4) Roles of Key Residues

Several positively charged (cationic) residues in the pore appear crucial for guiding and stabilizing chloride ions. Simultaneously, small kinks in certain helices may act as structural "hinges," allowing or blocking chloride passage.

How to Interpret These Results:

(1) Bridging a Major Gap: The study tackles the mismatch between static "closed" CFTR structures and their known open-channel function. Successfully capturing a stable open state in MD simulations is a significant step toward reconciling what cryo-EM data shows versus what physiological experiments have long told us.

(2) Strength in Multiple Replicas: Running many simulation repeats (rather than relying on a single trajectory) lends credibility. Only if a phenomenon is reproducible across multiple runs can it be considered robust.

(3) Consistency with Mutational Data: Observing that known functional hotspots (e.g., specific charged residues) play a key role in the new pore model further validates these findings.

Important Caveats and Limitations:

(1) Simulation Timescales vs. Biology
Even extended MD (on the microsecond scale) is still much faster, simpler, and more controlled than real cellular processes.

(2) Physiological existence of the penta-helical pore
Although the simulations and results are highly compelling, several factors leave open the possibility of a physiological open conformation differing from the observed penta-helical pore. These factors include ATP hydrolysis, interactions with physiological binding partners, the native membrane environment, and regions not modeled in the CFTR structures, such as the R domain. Most importantly, the transmembrane voltage is very high (500mV).

Bottom Line:

This work delivers a long-awaited, near-physiological view of CFTR's open conformation. It provides a foundational structure against which future experimental and computational studies can be compared. By demonstrating reliable chloride conduction and matching established biophysical data, these simulations bring us closer to understanding - and potentially targeting - CFTR's gating mechanism in health and disease. Readers should applaud the breakthroughs while recognizing that further exploration (including more complex in vitro and in vivo experiments) will still be necessary to capture the full dynamism of CFTR in the living cell environment.

Reviewer #4 (Public review):

Summary:

The structural mechanism of anion permeation through the open CFTR pore has remained unresolved and is subject to ongoing debate. That is because even in CFTR structures obtained under conditions that normally maximally activate the channel (phosphorylation + ATP + non-hydrolytic mutations + potentiator drugs) a bottleneck region in the pore, too narrow to allow passage of hydrated chloride ions, is observed.

The present study uses molecular dynamics (MD) simulations initiated from such "quasi-open" states to address local conformational dynamics of the pore. The authors conclude that the quasi-open structure stably relaxes to a fully open conformation on the sub-microsecond time scale. They provide a detailed analysis of this fully open structure and of the mechanism of chloride permeation. They conclude that two major exit pathways (a central and a peripheral) exist for chloride ions, and that the ions remain near-fully hydrated throughout the pore: chloride-protein interactions displace only 1-2 waters from the first solvation shell. Furthermore, the simulations provide some hints for conformational changes involved in gating.

Strengths:

The findings are interpreted in the context of the large body of published functional studies on CFTR permeation properties, and caveats are adequately discussed.

Weaknesses:

The conclusions on gating would benefit from further discussions. In particular, a fair comparison of the timescale at which channel gating happens, and that of the MD simulations would strengthen the manuscript.