Cryo-EM structures of Kv1.2 potassium channels, conducting and non-conducting

Reviewer #1 (Public Review):

In this manuscript by Wu et al., the authors present the high resolution cryoEM structures of the WT Kv1.2 voltage-gated potassium channel. Along with this structure the authors have solved several structures of mutants or experimental conditions relevant to the slow inactivation process that these channels undergo and which is not yet completely understood.

One of the main findings is the determination of the structure of a mutant (W366F) that is thought to correspond to the slow inactivated state. These experiments confirm results in similar mutants in different channels from Kv1.2 that indicate that inactivation is associated with an enlarged selectivity filter.

Another interesting structure is the complex of Kv1.2 with the pore blocking toxin Dendrotoxin 1. The results shown in the revised version indicate that the mechanism of block is similar to that of related blocking-toxins, in which a lysine residue penetrates in the pore. Surprisingly, in these new structures, the bound toxin results in a pore with empty external potassium binding sites.

The quality of the structural data presented in this revised manuscript is very high and allows for unambiguous assignment of side chains. The conclusions are supported by the data. This is an important contribution that should further our understanding of voltage-dependent potassium channel gating. In the revised version, the authors have addressed my previous specific comments, which are appended below.

(1) In the main text's reference to Figure 2d residues W18' and S22' are mentioned but are not labeled in the insets.

(2) On page 8 there is a discussion of how the two remaining K+ ions in binding sites S3 and S4 prevent permeation K+ in molecular dynamics. However, in Shaker, inactivated W434F channels can sporadically allow K+ permeation with normal single-channel conductance but very reduced open times and open probability at not very high voltages.

(3) The structures of WT in the absence of K+ shows a narrower selectivity filter, however Figure 4 does not convey this finding. In fact, the structure in Figure 4B is constructed in such an angle that it looks as if the carbonyl distances are increased, perhaps this should be fixed. Also, it is not clear how the distances between carbonyls given in the text on page 12 are measured. Is it between adjacent or kitty-corner subunits?

(4) It would be really interesting to know the authors opinion on the driving forces behind slow inactivation. For example, potassium flux seems to be necessary for channels to inactivate, which might indicate a local conformational change is the trigger for the main twisting events proposed here.

Reviewer #2 (Public Review):

Cryo_EM structures of the Kv1.2 channel in the open, inactivated, toxin complex and in Na+ are reported. The structures of the open and inactivated channels are merely confirmatory of previous reports. The structures of the dendrotoxin bound Kv1.2 and the channel in Na+ are new findings that will of interest to the general channel community.

Review of the resubmission:

I thank the authors for making the changes in their manuscript as suggested in the previous review. The changes in the figures and the additions to the text do improve the manuscript. The new findings from a further analysis of the toxin channel complex are welcome information on the mode of the binding of dendrotoxin.

A few minor concerns:
(1) Line 93-96, 352: I am not sure as to what is it the authors are referring to when they say NaK2P. It is either NaK or NaK2K. I don't think that it has been shown in the reference suggested that either of these channels change conformation based on the K+ concentration. Please check if there is a mistake and that the Nichols et. al. reference is what is being referred to.

(2) Line 365: In the study by Cabral et. al., Rb+ ions were observed by crystallography in the S1, S3 and S4 site, not the S2 site. Please correct.

Reviewer #3 (Public Review):

Wu et al. present cryo-EM structures of the potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and presumably sodium-bound states at 3.2 Å, 2.5 Å, 2.8 Å, and 2.9 Å. The work builds on a large body of structural work on Kv1.2 and related voltage-gated potassium channels. The manuscript presents a plethora of structural work, and the authors are commended on the breadth of the studies. The structural studies are well-executed. Although the findings are mostly confirmatory, they do add to the body of work on this and related channels. Notably, the authors present structures of DTx-bound Kv1.2 and of Kv1.2 in a low concentration of potassium (which may contain sodium ions bound within the selectivity filter). These two structures add considerable new information. The DTx structure has been markedly improved in the revised version and the authors arrive at well-founded conclusions regarding its mechanism of block. Regarding the Na+ structure, the authors claim that the structure with sodium has "zero" potassium - I caution them to make this claim. It is likely that some K+ persists in their sample and that some of the density in the "zero potassium" structure may be due to K+ rather than Na+. This can be clarified by revisions to the text and discussion. I do not think that any additional experiments are needed. Overall, the manuscript is well-written, a nice addition to the field, and a crowning achievement for the Sigworth lab.

Most of this reviewer's initial comments have been addressed in the revised manuscript. Some comments remain that could be addressed by revisions of the text.

Specific comments on the revised version:
Quotations indicate text in the manuscript.
(1) "While the VSD helices in Kv1.2s and the inactivated Kv1.2s-W17'F superimpose very well at the top (including the S4-S5 interface described above), there is a general twist of the helix bundle that yields an overall rotation of about 3o at the bottom of the VSD."

Comment: This seemed a bit confusing. I assume the authors aligned the complete structures - the differences they indicate seem to be slight VSD repositioning relative to the pore rather than differences between the VSD conformations themselves. The authors may wish to clarify. As they point out in the subsequent paragraph, the VSDs are known to be loosely associated with the pore.

(2) Comment: The modeling of DTx into the density is a major improvement in the revision. Figure 3 displays some interactions between the toxin and Kv1.2 - additional side views of the toxin and the channel might allow the reader to appreciate the interactions more fully. The overall fit of the toxin structure into the density is somewhat difficult to assess from the figure. (The authors might consider using ChimeraX to display density and model in this figure.)

(3) "We obtained the structure of Kv1.2s in a zero K+ solution, with all potassium replaced with sodium, and were surprised to find that it is little changed from the K+ bound structure, with an essentially identical selectivity filter conformation (Figure 4B and Figure 4-figure supplement 1)."

Comment: It should be noted in the manuscript that K+ and Na+ ions cannot be distinguished by the cryo-EM studies - the densities are indistinguishable. The authors are inferring that the observed density corresponds to Na+ because the protein was exchanged from K+ into Na+ on a gel filtration (SEC) column. It is likely that a small amount of K+ remains in the protein sample following SEC. I caution the authors to claim that there is zero K+ in solution without measuring the K+ content of the protein sample. Additionally, it should be considered that K+ may be present in the blotting paper used for cryo-EM grid preparation (our laboratory has noted, for example, a substantial amount of Ca2+ in blotting paper). The affinity of Kv1.2 for K+ has not been determined, to my knowledge - the authors note in the Discussion that the Shaker channel has "tight" binding for K+. It seems possible that some portion of the density in the selectivity filter could be due to residual K+. This caveat should be clearly stated in the main text and discussion. More extensive exchange into Na+, such as performing the entire protein purification in NaCl, or by dialysis (as performed for obtaining the structure of KcsA in low K+ by Y. Zhou et al. & Mackinnon 2001), would provide more convincing removal of K+, but I suspect that the Kv1.2 protein would not have sufficient biochemical stability without K+ to endure this treatment. One might argue that reduced biochemical stability in NaCl could be an indication that there was a meaningful amount of K+ in the final sample used for cryo-EM (or in the particles that were selected to yield the final high-resolution structure).

(4) Referring to the structure obtained in NaCl: "The ion occupancy is also similar, and we presume that Kv1.2 is a conducting channel in sodium solution."

Comment: Stating that "Kv1.2 is a conducting channel in sodium solution" and implying that conduction of Na+ is achieved by an analogous distribution of ion binding sites as observed for K+ are strong statements to make - and not justified by the experiments provided. Electrophysiology would be required to demonstrate that the channel conducts sodium in the absence of K+. More complete ionic exchange, better control of the ionic conditions (Na+ vs K+), and affinity measurements for K+ would be needed to determine the distribution of Na+ in the filter (as mentioned above). At minimum, the authors should revise and clarify what the intended meaning of the statement "we presume that Kv1.2 is a conducting channel in sodium solution". As mentioned above, it seems possible/likely that a portion of the density in the filter may be due to K+.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

In this manuscript by Wu et al., the authors present the high-resolution cryoEM structures of the WT Kv1.2 voltage-gated potassium channel. Along with this structure, the authors have solved several structures of mutants or experimental conditions relevant to the slow inactivation process that these channels undergo and which is not yet completely understood.

One of the main findings is the determination of the structure of a mutant (W366F) that is thought to correspond to the slow inactivated state. These experiments confirm results in similar mutants in different channels from Kv1.2 that indicate that inactivation is associated with an enlarged selectivity filter.

Another interesting structure is the complex of Kv1.2 with the pore-blocking toxin Dendrotoxin 1. The results show that the mechanism of the block is different from similar toxins, in which a lysine residue penetrates the pore deep enough to empty most external potassium binding sites.

The quality of the structural data presented in this manuscript is very high and allows for the unambiguous assignment of side chains. The conclusions are supported by the data. This is an important contribution that should further our understanding of voltagedependent potassium channel gating. Specific comments are appended below.

(1) In the mains text's reference to Figure 2d residues W18' and S22' are mentioned but are not labeled in the insets.

Now labeled in Fig. 2D

(2) On page 8 there is a discussion of how the two remaining K+ ions in binding sites S3 and S4 prevent permeation K+ in molecular dynamics. However, in Shaker, inactivated W434F channels can sporadically allow K+ permeation with normal single-channel conductance but very reduced open times and open probability at not very high voltages.

Addressed in the Discussion, lines 480-490.

(3) The structures of WT in the absence of K+ show a narrower selectivity filter, however, Figure 4 does not convey this finding. In fact, the structure in Figure 4B is constructed at such an angle that it looks as if the carbonyl distances are increased, perhaps this should be fixed. Also, it is not clear how the distances between carbonyls given in the text on page 12 are measured. Is it between adjacent or kitty-corner subunits?

We decided to remove mention of carbonyl distances, because at our resolutions the atoms are not resolved.

(4) It would be really interesting to know the authors' opinions on the driving forces behind slow inactivation. For example, potassium flux seems to be necessary for channels to inactivate, which might indicate a local conformational change is the trigger for the main twisting events proposed here.

We cite Sauer et al. (2011) for the idea that the intact selectivity filter is a strained conformation, and its relaxation yields the wide vestibule seen in NaK2K and Kv channels. Lines 434-439.

Reviewer #2 (Public Review):

There are four Kv1.2 channel structures reported: the open state, the C-type inactivated state, a dendrotoxin-bound state, and a structure in Na+.

A high-resolution crystal structure of the open state for a chimeric Kv1.2 channel was reported in 2007 and there is no new information provided by the cryoEM structure reported in this study.

The cryo-EM structure of the C-type inactivated state of the Kv1.2 channel was determined for a channel with the W to F substitution in the pore helix. A cryo-EM structure of the Shaker channel and a crystal structure of a chimeric Kv1.2 channel with an equivalent W to F mutation were reported in 2022. Cryo-EM structures of the C-type inactivated Kv1.3 channel are also available. All these previous structures have provided a relatively consistent structural view of the C-type inactivated state and there is no significant new information that is provided by the structure reported in this study.

A structure of the Kv1.2 channel blocked by dendrotoxin is reported. A crystal structure of charybdotoxin and the chimeric Kv1.2 channel was reported in 2013. Density for dendrotoxin could not be clearly resolved due to symmetry issues and so the definitive information from the structure is that dendrotoxin binds, similarly to charybdotoxin, at the mouth of the pore. A potential new finding is that there is a deeper penetration of the blocking Lys residue in dendrotoxin compared to charybdotoxin. It will however be necessary to use approaches to break the symmetry and resolve the electron density for the dendrotoxin molecule to support this claim and to make this structure significant.

We have now succeeded in breaking the symmetry and present in Fig. 3 a C1 structure of the toxin-channel complex. In the improved map we now see that our previous conclusion was wrong: the penetration of Lys5 cannot be much deeper than that seen in CTx and ShK structures. However for some reason the pattern of ion-site occupancies in the blocked state is different in this structure than in the others. Fig. 3, Fig. 4E; text lines 559-568.

The final structure reported is the structure of the Kv1.2 channel in K+ free conditions and with Na+ present. The structure of the KcsA channel by the MacKinnon group in 2001 showed a constricted filter and since then it has been falsely assumed by the K channel community that the lowering of K concentration leads to a construction of the selectivity filter. There have been structural studies on the MthK and the NaK2K channels showing a lack of constriction in the selectivity filter in the absence of K+. These results have been generally ignored and the misconception of filter constriction/collapse in the absence of K+ still persists. The structure of the Kv1.2 channel in Na+ provided a clear example that loss of K+ does not necessarily lead to filter constriction.

We are grateful to the reviewer for pointing out this serious omission. We now cite other work including from the Y. Jiang and C. Nichols labs showing examples of outer pore expansion and destabilization. Page p. 4, lines 90-104; lines 421-439.

The structure in Na+ is significant while the other structures are either merely reproductions of previous reports or are not resolved well enough to make any substantial claims.

We now state more clearly the confirmatory nature of our Kv1.2 open structure (lines 71-74) and the similarities of the inactivated-channel structures (lines 193196).

Reviewer #3 (Public Review):

Wu et al. present cryo-EM structures of the potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and presumably sodium-bound states at 3.2 Å, 2.5 Å, 2.8 Å, and 2.9 Å. The work builds on a large body of structural work on Kv1.2 and related voltage-gated potassium channels. The manuscript presents a large quantity of structural work on the Kv1.2 channel, and the authors should be commended on the breadth of the studies. The structural studies seem well-executed (this is hard to fully evaluate because the current manuscript is missing a data collection and refinement statistics table). The findings are mostly confirmatory, but they do add to the body of work on this and related channels. Notably, the authors present structures of DTXbound Kv1.2 and of Kv1.2 in a low concentration of potassium (with presumably sodium ions bound within the selectivity filter). These two structures add new information, but the studies seem somewhat underdeveloped - they would be strengthened by accompanying functional studies and further structural analyses. Overall, the manuscript is well-written and a nice addition to the field.

The data collection and refinement table has been added (Fig. 4 supplement 3.)

We agree and regret the lack of functional studies. We have not been able to carry them out because work in our laboratory is winding down and the lab soon will be closing.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

(1) It is not obvious from the data shown how well the side chain positions in the inactivated state are defined by the electron density. These figures should be redone. Maybe the use of stereo would be useful. This will be particularly useful for the reader to decide if the small changes in, for example, the positioning of the carbonyl oxygens are believable.

Figure 2 – figure supplement 4 shows the stereo views.

(2) The authors note the changes observed (though small) in the VSD which were not observed in other structures. The relevance of this observation is not described. Do these changes arise due to the different environments of detergents versus nanodisc etc. in the different structures?

We’ve now inserted a note about variety of environments and how this might be a cause of the difference: lines 280-285.

Are there changes in the pore-VSD interface in the inactivated and the open channel structures and if yes, then do mutations at these residues affect inactivation?

There is surprisingly little movement at the S4-S5 interface residues identified by Bassetto et al. (2022) as having effects on inactivation. Lines 262-267.

(3) For the structures in Na+, it is important to provide analytical data showing the biochemical behavior of the channel. This is also true for the wild type and the W to F mutant channel. Size exclusion profiles should be included.

The SEC profile (noisy, but showing a clear peak) of the channel in Na+ is now shown in Fig. 4 supplement 1. Low expression of the W366F mutant produced even worse SEC results, but we include a representative micrograph of W366F in Na+ to show the monodispersed protein prep. In Figure 5 – figure supplement 1.

Reviewer #3 (Recommendations For The Authors):

Portions of text from the manuscript are indicated by quotations.

Introduction: "One goal of the current study was to examine the structure of the native Kv1.2 channel."

Comment, minor points: The authors refer to the Kv1.2 construct used for the structural studies as "native Kv1.2". I found this somewhat confusing because the word "native" suggests derived from a native source. The phrasing above also gives the impression that the structure by Wu et al is the first structure of Kv1.2. The Kv1.2 construct is essentially identical to the one used by Long et al in 2005 to determine the initial structure of Kv1.2 (PDB 2A79). The authors discuss a subsequent paddle-chimera Kv1.2-2.1 structure from 2007 (PDB 2R9R) in the introduction, but it would be prudent to mention the 2005 one of Kv1.2 as well. The open structure determined by Wu et al. is an improvement on the 2A79 structure in that the 2A79 structure was modeled as a poly-alanine model within the voltage sensor domain. Nevertheless, the Kv1.2-2.1 structure (2R9R) is highly similar to the 2A79 structure of Kv1.2. The 2007 structure indicated that Kv1.2-2.1 recapitulates structural features of Kv1.2. It is therefore not surprising that the open structure presented here is highly similar to that of both PDB 2A79 (Kv1.2) and PDB 2R9R (Kv1.2-2.1).

We failed to point out the high quality of the original Long et al. 2005 structure and its comparisons with the chimeric structure in Long et al. 2007. We now have tried to correct this: lines 70-74.

Comment: The cryo-EM analyses suggest that a large percentage (most?) of the particles are missing the beta subunit. This should be commented on somewhere.

Now noted on lines 120-132, we pooled particles with and without beta subunits.

Regarding ions in the selectivity filter, one-dimensional plots of the density would strengthen the analysis.

Now included in Fig. 4.

Also, one should mention caveats associated with identifying ions in cryo-EM maps and the added difficulty/uncertainty when the density is located along a symmetry axis (C4 axis, due to the possible build-up of noise). C1 reconstructions, showing density within the filter, if possible, would strengthen the analyses.

You are correct. However local resolution is highest in the selectivity filter region. So I think that since the CTF-based filtering is constant over all the structure I think the SNR will be good on axis.

Comment: The section on channel inactivation could be simplified by stating that the structure is highly similar to W17'F structures of other Kv channels. (And then discussing possible differences).

We now note, “overall conformational difference is identical…” p. 7, lines 193-196.

"Salt bridges involving the S4 Arg and Lys residues are shifted slightly (Figure 2-figure supplement 3A-D). Arg300 (R3) is in close proximity to Glu226 on the S2 helix for the open channel, while R3 is closer to Glu183 in the S2 helix. The Glu226 side chain adopts a visible interaction with R4 in the inactivated state."

Comment: The density for these acidic amino acids seems weak, especially in the inactivated state. It seems like a stretch to make much of their possible conformational changes.

We’ve included stereo pairs in Fig. 2 – figure supplement 4.

"By adding 100 nM α-DTx to detergent solubilized Kv1.2 protein we obtained a cryo-EM structure at 2.8 Å resolution of the complex."

Comment: 100 nm. might be lower than the Kv concentration. The current methods are ambiguous on the concentration of Kv channel used for the DTx sample. From the methods, it seems possible that 100 nM DTX is a sub-stoichiometric amount relative to the channel. Regardless, the cryo-EM data seems to suggest that a large percentage of particles do not have DTx bound. This surely complicates the interpretation of density within the filter (which has partly been ascribed to a lysine side chain from DTx).

The reviewer correctly points a potentially serious problem. It turns out that the 100nM figure we quoted was incorrect, and the actual concentration of toxin, >400 nM, was substantially greater than the protein concentration. This is confirmed by the small fraction (<1%) of 3D class particles that do not show the toxin density (lines 303-306).

Comment: The methods on atomic structure building/refinement (Protein model building, refinement, and structural analysis) are sparse. A table is needed showing data collection and refinement statistics for each of the structures. This data should also provide average B factors for the ions in the filter. An example can be found in PMID 36224384.

Data collection and statistics are now in Fig. 4 – figure supplement 3.

"In the selectivity filter of the toxin-bound channel (Figure 3E) a continuous density is seen to extend downward from the external site IS0 through to the boundary between IS1 and IS2. This density is well modeled by an extended Lys side chain from the bound toxin, with the terminal amine coordinated by the carbonyls of G27”.

Comment: While there seems to be extra density in site IS0 from the figures, the density ascribed to lysine in the filter doesn't seem that distinct from those of ions in the open structure. 1-dimensional density plots and some degree of caution may be prudent. Could there, for example, be a mixture of toxin-bound and free channels in the dataset?

Could the lysine penetrate to different depths? If the toxin binds with nM affinity, why are any channels missing the toxin? Have the authors modeled an atomic structure of the entire toxin bound to the channel to evaluate how plausible the proposed binding of the lysine is? Can the toxin be docked onto Kv1.2 with the deep positioning of the lysine and not clash with the extracellular surface of Kv1.2?

We also were concerned about these issues. We have been able to obtain a C1 reconstruction of the toxin-channel complex. In building the atomic model we found that indeed the Lys5 side chain could not penetrate as far as we had thought, and appears to be coordinated by the first carbonyl pair. Fig. 3; text lines 331-332.

"Toxin binding shrinks the distances between opposing carbonyl oxygens in the selectivity filter, forming a narrower tunnel into which the Lys side chain fits (Figure 3F). The second and fourth carbonyl oxygen distances are substantially reduced from 4.7 Å and 4.6 Å in an open state to 3.7 Å and 3.9 Å, respectively (Figure 4E). In a superposition of Kv1.2 open-state and α-DTX-bound P-loop structures, there is also an upward shift of the first three carbonyl groups by 0.7~1.0 Å (Figure 4F). "

Comment: I suspect the authors intend to refer to Figure 3F rather than 4. I would be cautious here. The refined positions of the carbonyl oxygens are almost certainly affected by the presence or absence of ions in the atomic model during refinement. The density and the resolution of the map may not be able to distinguish small changes to the positions of the carbonyl oxygens (and these differences/uncertainties are compounded by the C4 symmetry).

"On the other hand, the terminal amine of lysine in α-DTX is deeply wedged at the second set of carbonyls, narrowing both IS1 and IS2 while displacing ions from the sites (Figure 3-figure supplement 2A). CTX does not cause narrowing of the selectivity filter or displacements of the carbonyls (Figure 3-figure supplement 2B). "

Comment: Again, caution would be prudent here.

We are very grateful to the reviewer for pointing out these problems. We have removed these statements that are weakly supported at our resolution level.

"Shaker channels are able to conduct Na+ in the absence of K+ (Melishchuk et al., 1998)."

Comment: How about the Kv1.2 channel? Is Kv1.2 able to conduct Na+ in the absence of K+ ? This would certainly be relevant for interpreting the conformation of the filter and the density ascribed to Na+ for the structure in sodium.

We agree wholeheartedly, but unfortunately we are no longer capable of doing the measurements as our lab will soon close.

"Ion densities are seen in the IS1, IS3, and IS4 ion binding sites, but the selectivity filter shows a general narrowing as would be expected for binding of sodium ions. The second, third, and fourth carbonyl oxygen distances are reduced from 4.7 Å, 4.7 Å, and 4.6 Å in the open state to 4.4 Å, 3.9 Å, and 4.5 Å, respectively. The rest of the channel structure is very little perturbed. "

Comment: The density for IS4 seems weak. To me, it looks like IS1 and IS3 are occupied, whereas IS2 and IS4 are much weaker. 1-dimensional density plots would be helpful. I would suggest caution in commenting too strongly on the "general narrowing" since the resolution of the maps, the local density, and the atomic structure refinement would be consistent with coordinate errors of 0.5 Å or more - and would be compounded (~ doubled) by measuring between symmetry-related atoms.

We present 1D plots in Fig. 4E. We no longer comment on “narrowing”

"Finally, the snake toxin a-Dendrotoxin (DTx) studied here is seen to block Kv1.2 by insertion of a lysine residue into the pore."

Comment: Discussion (and references) should be given regarding what was known prior to this study on the mode of inhibition by DTx.

Discussion and references now added, lines 287-301.

"On the other hand, a lengthy molecular-dynamics simulation of deactivation in the Kv1.2-2.1..."

Comment: I don't think mentioning this personal communication adds to the manuscript.

Actually the original “personal communication” reference was there because the situation is complicated. The movie S3 accompanying the Jensen et al. paper shows deactivation and dewetting of the channel during a 250 us simulation. In the movie there are ions visible in the selectivity filter for the first 50 us, but after that the SF appears empty. Puzzled by this we contacted Dr. Jensen who explained that the movie was in error, ions remain in the SF throughout the entire 250 us. We now cite Jensen (2012) along with the personal communication.

"The difference between the open and inactivated Kv1.2 structures, like the difference in Kv1.2-2.1 (Reddi et al., 2022) and Shaker (Tan et al., 2022) can be imagined as resulting from a two-step process."

Comment: Confusing phrasing because the authors mean to compare their structure to inactivated structures of Kv1.2-2.1 and shaker.

Fixed, lines 220-222.

"Molecular dynamics simulations by Tan et al. based on the Shaker-W17'F structure show that IS3 and IS4 are simultaneously occupied by K+ ions in the inactivated state."

Comment: I think that the word "show" is too strong. Perhaps "suggest"

The MD result seems to us to be unequivocal, that most of the time the two sites are occupied by ions.

References are needed for the following statements:

- "as well as the charge-transfer center phenylalanine"

Now citing Tao et al. 2010, line 156.

- "total gating charge movement in Shaker channels is larger, about 13 elementary charges per channel"

Now citing the review by Islas, 2015 (line 166-169).

"The selectivity filter of potassium channels consists of an array of four copies of the extended loop (the P-loop) formed by a highly conserved sequence, in this case, TTVGYGD. Two residues anchor the outer half of the selectivity filter and are particularly important in inactivation mechanisms (Figure 2B, right panels). Normally, the tyrosine Y28' (Y377 in Kv1.2) is constrained by hydrogen bonds to residues in the pore helix and helix S6 and is key to the conformation of the selectivity filter. The final aspartate of the P-loop, D30' (D379 in Kv1.2) is normally located near the extracellular surface and has a side chain that also participates in H-bonds with W17' (W366 in Kv1.2) on the pore helix."

Citations added (Pless 2013, Sauer 2011) lines 211-214.

- "During normal conduction, ion binding sites in the selectivity filter are usually occupied by K+ and water molecules in alternation."

Added Morais-Cabral et al. 2001, p. 17, lines 463-465.