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 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 voltage-dependent 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.

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+ 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?

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.

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.

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.
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.

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 DTX-bound 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.