Peer review process
Consolidated peer review report (5 September 2023)
GENERAL ASSESSMENT
KATP is a remarkably important potassium-selective ion channel that is inhibited by intracellular ATP, allowing it to serve key roles throughout the body, including regulation of insulin release from the pancreas. Driggers et al. describe an important study that reports the structure of an open KATP channel complex formed from the Q52R diabetes mutation of Kir 6.2, SUR1, and long-chain PIP2. Although earlier structures have been determined in closed, open and inactivated conformations, none have resolved where PIP2 binds. This has been an important limitation of the available structures given the key role of this membrane component in promoting the open state and influencing the inhibitory actions of intracellular ATP. The open channel structure reported here resembles a previous structure from the MacKinnon laboratory of a mutant channel that exhibits a very high open probability (G334D, C166S). The authors also show that channel opening is accompanied by a dilation of the Kir6.2 inner helix gate, in particular rotation of L164 and F168 away from the channel's pore, and by a conformational rearrangement at the interface between Kir6.2 and the TMD0 domain of SUR1.
The key advance that allowed the authors to resolve PIP2 bound to KATP appears to be the use of brain-derived long-chain PIP2, which was incubated with membranes prior to extraction of the channel protein with digitonin and purification, as well as the use of the Q52R mutant that promotes the open state. Remarkably, the structures of PIP2 bound to KATP reveals two PIP2 binding sites: one related to previously resolved sites in other Kir channels; and a new, unanticipated one where the lipid snuggles into the interface between Kir6.2 and the TMD0 helix of SUR1, a region of SUR1 previously implicated in promoting the open state of KATP. To examine the functional impact of the two PIP2 binding sites, the authors identify two positively-charged residues – one in each binding site – to neutralise by mutation to alanine, in an effort to disrupt the two putative PIP2 interactions. Indeed, they find that KATP channel currents on patch excision are very small and require much longer exposure to PIP2 to fully activate, compared to wild-type channels. Although one could question the physiological relevance of the new PIP2 binding site, that PIP2 remains bound throughout extraction and purification in digitonin solutions, and can be readily resolved in the structure, suggests that interactions of longer-chain PIP2 with both sites is quite favourable and likely to be occupied under biological conditions. One of the remarkable features of the new structures is that PIP2 binding to KATP causes a conformation change in the inhibitory ATP binding site, providing a mechanistic explanation for how PIP2 and ATP antagonistically engage to promote open or closed states, respectively. The structure also reveals how the Q52R mutant likely forms a cation-π interaction with W51 of SUR1, explaining how the diabetes mutation promotes the open state of KATP. Ultimately, further experiments will help unravel the physiological impact of the newly identified PIP2 site, as the electrophysiology presented in this structural study is understandably limited. However, that does not diminish the impact of the study.
Overall, this is an important study that helps to explain how PIP2 exerts such a profound regulatory influence over KATP, which will be valuable for future studies on KATP and of general interest to scientists investigating how PIP2 regulates other membrane proteins. The preprint is well-written, the work appears to have been carried out with rigor and attention to detail, and the authors present new conclusions and discuss them in the context of previous findings, some of which have been enigmatic until now.
RECOMMENDATIONS
Essential revisions:
Most of the figures focus on a comparison of the new PIP2-bound open state for the Q52R mutant with a closed state of KATP. A more extensive structural comparison between the PIP2-bound open structure and other open structures solved in absence of PIP2 (e.g. ref 27 where Kir6.2 mutations C166S and G334D were used) would help clarify the functional roles of the putative PIP2 binding sites. A structural comparison of the PIP2 binding sites between the two open structures (apo and holo) would reveal whether the PIP2 binding sites are conserved in the absence of PIP2, for example. Such a comparison would help the general reader to understand which of the structural changes observed in the new structure have been seen before and those that have not. Another example would be to clarify the extent to which structural changes around the inhibitory ATP binding site seen here are related to those in previous structures thought to be open.
It would be valuable for readers if the authors could explain their thinking about why the new PIP2 binding site is likely to be physiologically relevant. In its current form, some readers may be unconvinced about whether the new site is occupied under physiological conditions. For example, in the last paragraph of page 15, the authors acknowledge that in their previously published closed structures without exogenous PIP2, they saw lipid densities in the novel PIP2 site which they modelled as phosphatidylserines. Similar lipid densities were also seen near this site in the other published open state (see Fig. S6 in DOI:10.1073/pnas.2112267118). In the next paragraph on page 16, they also comment on the unusually low specificity of Kir6.2 towards phosphoinositides and other lipids, and the ability of purified KATP channels to open in the absence of PIP2. Given these findings, and the potentially high concentration of PIP2 incubated with the sample, is it conceivable that the new PIP2 site is not occupied under physiological conditions? What do the authors know about the molar fraction of PIP2 achieved in the final sample and how this might compare to the estimates of PIP2 abundance in native human cell membranes (0.2-1%)? Would PIP2 ever reach high enough concentrations in the membrane for this site to be bound? The authors might also emphasize that the channel was only exposed to brain PIP2 for a short time before being extracted and purified in the detergent solution, indicating that the interaction between PIP2 and the channel at both sites is quite strong and likely to be occupied physiologically.
The electrophysiological data presented in Fig. 2, while corroborating the existence of a second PIP2 site, is not definitive. On page 9 of the text, the authors mention striking differences between wild-type and mutant channels in terms of "the initial currents upon membrane excision, the extent of current increase upon PIP2 stimulation, and the PIP2 exposure time required for currents to reach maximum." The extent of current increase is shown for multiple patches in Fig. 2E and the other differences are inferred from representative traces. The authors may wish to include some form of quantification for the amount of initial current and time course for data from multiple patches. For example, the authors mention "barely detectable currents" for the SUR1-K134A/Kir6.2-R176A mutant. Taking into account the difference in scale bars, the currents in the example provided don't look any smaller than the currents from Kir6.2-R176A/SUR1 channels. Given the proximity between the two sites, it seems possible that a mutation in one site could allosterically affect PIP2 binding at the other site. In principle, two mutations could independently affect PIP2 binding at the same functional site and have additive effects. Perhaps the strongest arguments in favour of two distinct functional sites come from the mutation map in Fig. 7, which nicely matches the two bound PIP2 molecules, and previous studies showing that KATP is less sensitive to PIP2 in the absence of SUR1, which forms part of the second binding site.
The increase in current in PIP2 in Fig. 2E may represent the extent of the increase in probability of opening. However, calculating the fold increase in current depends on accurate measurements of the very small currents at the beginning of the experiment, which will be heavily affected by residual leak or noise. In the absence of any direct measurements of open probability (for example with single channels), the authors may wish to discuss these limitations in the text.
Optional suggestions:
The Kir6.2-Q52R mutation, which stabilizes the open channel, mediates its effects by interacting with W51 on SUR1 (Fig. 6). Q52R is also very close to the PIP2 headgroup in the novel binding site and thus could help stabilize PIP2. Hence, it would be interesting to test the PIP2 sensitivity of this mutant in excised patches, as in Fig. 2D,E. If PIP2 binding at the second site is favoured by the mutation, the PIP2-induced increase of KATP current should be lower than that observed in WT channels.
It would be helpful for the authors to provide a biochemical interpretation for the functional results in Fig. 2 D, E. It would seem that the two mutations, Kir6.2-H176A and SUR1-K134A, diminish PIP2 binding affinity but do not prevent PIP2 binding, as the low basal currents seen in the mutants can be rescued by increasing PIP2 concentration.
The discussion on the role of H175 on pH regulation is interesting, but speculative. As the H175K mutant still undergoes acid-induced inhibition (ref 39), it does not seem appropriate to state that the H175K mutant "abolishes channel sensitivity to pH" (p7). A positive charge at position H175 increases basal activity, suggesting that the cationic form of H175 mediates acid-induced activation. The structure shows two H175 rotamers but does not clarify which of these rotamers are populated at different pH values. In addition, there is no evidence to suggest that the cationic form of H175 preferentially interacts with PIP2 and that its neutral form interacts with E179 (p8). On the contrary, it seems more logical to predict that the cationic form of H175, which is positively charged, interacts with E179, which is negatively charged. It would be helpful to clarify several of these points.
4)There are many questions that come to mind that might be interesting topics to add to the discussion. What is the relative affinity of this novel site for PIP2 over other PI's and even other lipids? Given that previous attempts to establish this (e.g. cited in reference 38) may have been measuring the summed contribution of both sites, if both are functionally relevant (as the present results suggest), do the sites differ in their selectivity? Could this be a lipid binding pocket, which has been displaced by high levels of PIP2 and a locked-open channel? These are not trivial questions to answer, but they are important for understanding the relative importance of the two PIP2 binding sites for the function of KATP and it would be useful to discuss the limits of what can be reasonably concluded at this time. Some of these points might be addressed in the results while other could add to the discussion. In thinking about the roles of the two PIP2 binding sites, have the authors considered the possibility that the PIP2 site found in other Kir channels might act as a reservoir for PIP2 and that PIP2 moves to the new site at the interface with SUR1 once the channel opens?
Page 9, lines 6-10: The authors suggest that the slower washout of long-chain PIP2 activation from excised patches compared to that of short-chain synthetic PIP2 is due to hydrophobic interactions between the longer acyl chains and KATP. However, this observation has been previously explained by the differences in the solubility of short- and long-chain PIP2 and therefore their rate of partition into and out of the plasma membrane. Is any data available to distinguish these possibilities?
Can the authors provide higher quality micrographs in Fig. S1 along with a scale bar. Why are three different micrographs shown? Also, this figure would probably benefit from moving some of the text embedded in the figure to a traditional legend along with a somewhat expanded description of what is shown graphically in the figure.
In the main text when describing the results in Fig. 2D, it would be helpful for the general reader to first explain the protocol employing both low and high ATP concentrations and what value this has for assessing the impact of mutations. As it currently stands, the reader is left guessing why this expertly devised protocol was used.
In Fig. 3 it would be helpful to align the three panels so the reader can appreciate how the structure gives rise to the pore radius plot in panel C. Also, the point made about the G-loop not changing appreciably between closed and opens states would be good to show in the structures.
The G-loop was previously proposed to aid in preventing the leakage of K ions into the internal solution as polyamines block Kir channels (Xu et al, 2009 NSMB). It might be worth commenting on this as it seems compatible with what is found here in that region.
Fig. 4A could be improved. The superimposition of open and closed structures in panel A takes some time for the reader to grasp. Maybe showing structures side by side with key distance measurements highlighting regions where there is movement between open and closed states would help, and then showing superimposition for a more limited view of where PIP2 binds? In panels B and C, it is not easy to appreciate how the structure in the open state disrupts the binding of ATP to the inhibitory site. Perhaps some use of space-filling models like those in Fig. S6 would help to illuminate the space occupied by ATP in the closed state, along with a zoomed-in view of all the residues coordinating ATP, and also similar views for how the conformational change during opening would interfere with ATP binding or move key coordinating residues. Fig. 4 contains a lot of information but it is not presented in a way that is easy for the reader to comprehend.
In the figures, the authors focus their comparisons between the structure solved in this manuscript (open, PIP2 bound) and previous structures solved in the same lab (closed, ATP and/or inhibitors bound). While comparisons are made in the text to the open and 'pre-open' structures solved by other investigators, it might be clearer if visual comparisons were offered as well – especially of the interaction between the SUR1-W51 residue and the wild-type Kir6.2-Q52 residue in both other structures, the similarity of which offers support for the authors arguments about common structural rearrangements on page 17.
Could the authors comment on how the Rb efflux assay results in Fig. 6 panel D add to the electrophysiological results shown in that figure in panels B and C? Differences in data from the flux assay in Fig. 6D may reflect changes in channel function, but they may simply reflect different expression levels for mutant channels.
The map in Fig. 7 corresponds to both loss-of-function mutations, that cause diabetes, and gain-of-function mutations, that cause hyperinsulinism. Is it the opinion of the authors that these mutations mediate their effects by modulating PIP2 binding? LOF mutations could reduce PIP2 binding whereas GOF mutations could strengthen PIP2 binding.
As referred to above, Fig. S6 in DOI:10.1073/pnas.2112267118 shows lipid densities near the new PIP2 site – how do they compare to the location of the PIP2 densities resolved in this manuscript? Are the lipid densities present in Fig. S3C and D also compatible with PC?
The idea advanced in the discussion and Fig. S6 that PIP2 binds to the new site only after the channel opens is interesting and seems conceptually related to what was recently proposed for PIP2 modulation of KCNQ by Mandala and MacKinnon (PNAS 2023). It might be helpful for the reader to see those dots connected.
The allosteric models of ligand regulation of the KATP channel have been predicated on the existence of four PIP2 binding sites across the molecule – how does the existence of eight potential PIP2 binding sites alter previous attempts to quantitively model KATP activity (e.g. reviewed in DOI:10.1085/jgp.200308878 and DOI:10.1085/jgp.201711978)? Perhaps this deserves a comment.
The experiments described on pages 13-14 and ion Fig. 6 that explore the Kir6.2-Q52 and SUR1-W51 interaction are convincing, but the dose-response curves (especially for WT and the W51C-Q52R) would benefit from some lower concentrations of ATP.
REVIEWING TEAM
Reviewed by:
Surbhi Dhingra, Postdoctoral Fellow, NINDS, NIH, USA: structural biology (cryo-electron microscopy) and ion channel mechanisms
Jerome Lacroix, Associate Professor, Western University of Health Sciences: ion channel mechanisms, electrophysiology, fluorescence spectroscopy
Michael C. Puljung, Assistant Professor, Trinity College, Hartford, CT, USA: ion channel mechanisms, electrophysiology, fluorescence spectroscopy
Xiaofeng Tan, Research Fellow, NINDS, NIH, USA: structural biology (X-ray crystallography and cryo-electron microscopy) and ion channel mechanisms
Samuel Usher, Postdoctoral Fellow, University of Copenhagen, Denmark: ion channel mechanisms, electrophysiology
Kenton J. Swartz, Senior Investigator, NINDS, NIH, USA: ion channel structure and mechanisms, chemical biology and biophysics, electrophysiology and fluorescence spectroscopy
Curated by:
Kenton J. Swartz, Senior Investigator, NINDS, NIH, USA
(This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Comments concerning minor and presentational issues have been omitted for brevity.)
