Structure of an open K _ATP channel reveals tandem PIP ₂ binding sites mediating the Kir6.2 and SUR1 regulatory interface

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1. 

   Biophysics Colab

   Jan 12, 2024

   Evaluation statement (10 January 2024)

   Driggers et al. is an elegant study that reports the structure of an open KATP channel complex formed from the Q52R diabetes mutation of the pore-forming subunit Kir 6.2, the sulfonylurea receptor (SUR1), and long-chain phosphatidylinositol 4,5-bisphosphate (PIP2) – a key lipid that stabilizes the open state of the channel and regulates inhibition by intracellular ATP. The structure reveals one PIP2 site related to that seen in other Kir channels as well as a second unanticipated one where the lipid snuggles into the interface between Kir6.2 and a region of SUR1 previously implicated in promoting the open state of KATP. This important finding helps to explain how PIP2 exerts such a profound regulatory influence on KATP.

   Biophysics Colab considers this to be a convincing study and recommends it to … More

   Evaluation statement (10 January 2024)

   Driggers et al. is an elegant study that reports the structure of an open KATP channel complex formed from the Q52R diabetes mutation of the pore-forming subunit Kir 6.2, the sulfonylurea receptor (SUR1), and long-chain phosphatidylinositol 4,5-bisphosphate (PIP2) – a key lipid that stabilizes the open state of the channel and regulates inhibition by intracellular ATP. The structure reveals one PIP2 site related to that seen in other Kir channels as well as a second unanticipated one where the lipid snuggles into the interface between Kir6.2 and a region of SUR1 previously implicated in promoting the open state of KATP. This important finding helps to explain how PIP2 exerts such a profound regulatory influence on KATP.

   Biophysics Colab considers this to be a convincing study and recommends it to scientists working on KATP and other membrane proteins regulated by PIP2.

   (This evaluation by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

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2. 

   Biophysics Colab

   Jan 12, 2024

   Authors' response (22 December 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 …

   More

   Authors' response (22 December 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.

   We thank the reviewers for the positive evaluation of our work and helpful suggestions. We have revised the manuscript to address the essential revisions as well as additional suggestions from the reviewers. Moreover, since the preprint was posted in bioRxiv, we have conducted additional analysis of our cryoEM data and identified a closed channel conformation in which the new PIP2 binding site is occluded and the cytoplasmic domain of Kir6.2Q52R is rotated and translated away from the plasma membrane--a conformation that corresponds to an apo SUR1/Kir6.2Q52R and is similar to our previously published WT apo (no PIP2 added) closed structure. We have also performed additional mutational and functional studies to show the dependence of the gain-of-function phenotype of Kir6.2Q52R on intact PIP2 binding sites. The additional data included in the revised manuscript further strengthens the conclusions of our study.

   Below we provide detailed response to the reviewers' essential revision requests and additional suggestions.

   RECOMMENDATIONS

   Essential revisions:

   1. 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.

   We thank the reviewers for the excellent suggestion to compare the PIP2-bound SUR1/Kir6.2Q52R structure with previous open KATP channel structures.. We have now included a comparison of the PIP2-bound SUR1/Kir6.2Q52R structure (holo) and the previously published pre-open structure (rodent SUR1-39aa-Kir6.2H175K fusion channel; PMID: 35589716) in the revised Figure S4. Note that while the open structure of human SUR1/Kir6.2C166S, G334D (PMID: 34815345) is also very similar to our PIP2-bound SUR1/Kir6.2Q52R structure, due to sequence variations in the different species of SUR1 used and an additional amino acid present in the construct (glycine at aa position 2), we have chosen to compare our structure with the pre-open rodent structure to avoid confusion in key SUR1 residues shown. In revised Fig.S4a,b, we also compared lipid density in the PIP2 binding pocket of all three open structures to highlight the difference between the long-chain PIP2 in this study, and the unassigned density in the pocket observed in the open structure of human SUR1/Kir6.2C166S, G334D (no exogenous PIP2 added) and the pre-open structure of rodent SUR1-39aa-Kir6.2H175K fusion (short-chain diC8-PIP2 added post purification of channel-containing detergent micelles).

   1. 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.

   We thank the reviewers for asking an important question about the physiological relevance of the two PIP2 binding sites. We addressed this question by introducing PIP2 binding site mutations in KATP channels expressed in native membranes, and tested the consequences of these mutations on channel activity (results shown in the revised Fig. 3). Mutating key residues involved in PIP2 binding, at either sites or both sites, markedly reduced channel activity in native membranes without exogenous PIP2. Adding exogenous PIP2 reversed the effects of these PIP2 binding-site mutations, providing strong evidence that these PIP2 binding sites have physiological roles in channel gating.

   Regarding the question of whether PIP2 binds at the novel binding site under physiological conditions, our structural analysis suggests it is likely state-dependent. When the channel is in the open conformation, the new site accommodates PIP2 as shown in our PIP2-bound structure. Although other phosphoinositides also stimulate channel activity when applied to isolated membrane patches containing channels, given PI(4,5)P2 is the most abundant phosphoinositide in the plasma membrane, we suspect the site is likely bound to PI(4,5)P2 under physiological conditions. To address the frequency of PIP2 occupancy at the new PIP2 binding site under different physiological conditions (high ATP/ADP resembling high glucose, versus low ATP/ADP resembling low glucose) experimentally, alternative approaches will be needed, for example, by crosslinking channel subunits with bound lipids followed by identification with mass spectrometry, or solving channel structures in native membranes. These are studies we are very interested in pursuing.

   In the current study, we were interested in maximizing the chance of capturing channels in the PIP2-bound conformation to understand the structural basis of channel activation by PIP2. Even under the experimental conditions of exogenous PIP2, we did observe conformations that likely correspond to apo structure at the new PIP2 binding site (from our additional analysis of the cryoEM data; see revised Figure S2 and S8), indicating the new PIP2 binding site is dynamic and can bind PIP2 when the channel is open, but not when the channel is closed.Although we did not attempt to compare PIP2 abundance (molar fraction) before and after incubation with exogenous PIP2 in the current study, we have previously quantified PIP2 in cell membranes from control INS-1 (a rat insulinoma) cells and INS-1 cells overexpressing PIP5 kinase and labelled with 32P using thin-layer chromatography (Lin et al., Diabetes, 2005; PMID: 16186385). The study showed a ~30-fold increase in PIP2, which was accompanied by a ~26-fold decrease in ATP sensitivity of KATP channels measured by inside-out patch-clamp recording, and a decrease in glucose-stimulated insulin secretion (GSIS) response. While the study does not directly answer whether physiological PIP2 concentrations would ever reach high enough as our in vitro experimental system, it does show that increasing PIP2 levels by manipulating an enzyme involved in PIP2 synthesis can induce abnormally high channel activity (i.e. ATP-insensitive) and reduce GSIS, resembling phenotype caused by the neonatal diabetes mutation Kir6.2 Q52R that stabilizes the second PIP2 binding site.

   1. 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.

   We thank the reviewers for this great suggestion to include quantification of initial currents and response time to maximum currents after PIP2 exposure, which is now included in the revised Figure 3.

   1. 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.

   The initial currents were calculated as currents observed in K-INT solution upon patch excision minus currents measured in 1mM ATP (KATP currents are inhibited >99% at 1 mM ATP). As such, any leak currents were accounted for. We have now stated this in the electrophysiology section in Methods (page 15). For PIP2 stimulation experiments, PIP2 effects plateau with regard to channel P__o (i.e. total currents) and any further stimulatory effects are reflected by a gradual loss of ATP inhibition (which we check by exposure to 0.1mM or 1mM ATP as shown in revised Fig. 3). Patches that do not show the PIP2 plateauing effects on currents indicate potential leak, which we can confirm by exposing channels to the high affinity inhibitor glibenclamide. Patches showing significant leaks (gradual shift in baseline) were not included in the analysis.

   Optional suggestions:

   We appreciate the many excellent suggestions from the reviewers, and have made substantial changes in the revised manuscript based on these suggestions. They are detailed below:

   1. 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.

   From the angle shown in the original Fig.6A, Q52R does appear very close to the headgroup of PIP2 in the novel binding site. However, Q52R is actually very far away from the second bound PIP2. We have now provided additional viewing angle in the revised Fig. 7a that offers better visualization of the distance to avoid confusion.

   1. 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 text has been revised to better reflect the functional data (original Fig.2D,E, now shown in the revised Figure 3). It now reads (page 6 top): "To probe the functional role of the two PIP2 binding sites, we compared the PIP2 response of WT channels to channels containing the following mutations: Kir6.2R176A , which is predicted to weaken the first PIP2 binding site; SUR1K134A, which is expected to weaken the second PIP2 binding site; Kir6.2R176A and SUR1K134A, which weaken both PIP2 binding sites (Fig.3a)."

   1. 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.

   We thank the reviewers for the constructive criticisms and agree our statement is too speculative in the absence of experimental evidence that the cationic form of H175 preferentially interacts with PIP2 while its neutral form interacts with E179. Accordingly, we have removed discussion on the role of H175 and instead, simply describe what we observe in our structure.

   The revised paragraph now reads (page 5 top):

   Interestingly, the Kir6.2-H175 sidechain exhibited two rotameric positions in the PIP2-bound SUR1/Kir6.2Q52R channel cryoEM density map: one oriented towards PIP2 in the conserved binding site and the other towards E179 in the same Kir6.2 subunit (Fig.2b). Kir6.2-H175 has been previously implicated in acid-induced activation of KATP channels between pH 7.4 and 6.8 with a pK of 7.16. Mutating Kir6.2-H175 to lysine, which mimics protonated state of H175, increased basal channel activity and abolished channel activation by pH, suggesting protonation of H175 favors channel opening. Moreover, mutating Kir6.2-E179 to Q greatly attenuated pH-induced channel activation, suggesting Kir6.2-E179 also has a role in acid activation of KATP channels. Our structural observation that H175 sidechain has interaction with PIP2 head group in the conserved site and with Kir6.2-E179 is consistent with the aforementioned mutation-function correlation studies. Since our protein sample was prepared at pH~7.5 (see Methods), which predicts H175 to be largely unprotonated based on the pK of acid activation of the channel of 7.16, it remains to be determined how protonation of H175 at lower pH that activates the channel alters interaction with PIP2 and E179 to stabilize channel opening.

   1. 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?

   We agree that the many topics raised by the reviewers are all very interesting and worth pursuing in the future. We have tried to include these and point out limitations of our data in relation to these questions in the results and discussion. It is our hope that the reviewers' comments will spark interest for further research related to these topics. For example, we plan to use MD simulation studies to address whether PIP2 bound at the conserved site may migrate to the novel site.

   1. 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?

   We do not currently have data to distinguish these possibilities, but based on the structure it would be possible in the future to design mutations that perturb channel interaction with the acyl chains to dissect these possibilities.

   1. 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.

   The different micrographs with KATP particles are included to show the different areas of the grid coated with graphene oxide. In one area, the folds of the graphene oxide layer are clearly visible.

   1. 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.

   We have revised the text to better explain the rationale of the recording protocol in both the Results section and Methods. Specifically, the alternating brief exposures to low and high ATP were designed to monitor the gradual decrease of ATP sensitivity during PIP2 exposure.

   1. 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.

   We have revised this figure (now Fig. 4) according to the suggestion.

   1. 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.

   Since we do not have any direct experiments to test this, we have decided to leave out the reference in the current study.

   1. 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.

   We have revised this figure (revised Fig. 5) as suggested and hope it is now improved.

   1. 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.

   We have added a supplement figure (Fig. S6) to make this comparison.

   1. 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 Rb efflux data shown in the original Fig. 6 (now Fig. 7) complement the electrophysiology data and show channel behaviour in intact cells. For all mutants we confirmed comparable expression levels in transfected cells by Western blots.

   1. 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.

   We believe the reviewers meant loss-of-function mutations that cause hyperinsulinism and gain-of-function mutations that cause neonatal diabetes. As stated in the Discussion section, these mutations could exert their effects by modulating PIP2 binding or by affecting PIP2 gating allosterically.

   1. 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?

   Please see the comparison we have now included in the revised Fig.S4a, b.

   1. 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.

   We thank the reviewers for the suggestion and have now cited the paper by Mandala and MacKinnon (page 10 top, reference 49).

   1. 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.

   This is an interesting question that would be an excellent topic for researchers who are interested in kinetic modeling.

   1. 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.

   We have conducted additional experiments to obtain data at lower ATP concentrations (see revised Fig. 7).

   (This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

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   Read the original source
3. 

   Biophysics Colab

   Jan 12, 2024

   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 … More

   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:

   1. 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.

   2. 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.

   3. 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.

   4. 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:

   1. 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.

   2. 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.

   3. 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?

   1. 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?

   2. 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.

   3. 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.

   4. 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.

   5. 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.

   6. 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.

   7. 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.

   8. 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.

   9. 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.

   10. 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?

   11. 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.

   12. 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.

   13. 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.)

   

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   Version published to 10.1101/2023.08.01.551546v2 on bioRxiv

   Jan 11, 2024
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   Version published to 10.1101/2023.08.01.551546v1 on bioRxiv

   Aug 1, 2023