Aedes albopictus females looking for oviposition sites. The Flight Range of 90% of the mosquito population (FR90) was found to exceed 200 m, consistently with data obtained from a previous study conducted in a highly urbanised area in Rome . Modelling results showed that dispersal can be so rapid that insecticide spraying within a 200m-radius around a potential infected case leaves >10% probability that a potentially infected mosquito escapes the treatment, even if this is carried out after only 2–3 days since the importation of a viremic case. These data provide evidence in favour of an update of guidelines for the control of exotic autochthonous arbovirus transmission in temperate areas and highlight the need of effective surveillance approaches and rapid response to contain the risks associated to imported viremic cases.The increasing number of exotic arbovirus cases imported in Europe and the 2017 chikungunya outbreak in central/southern Italy highlight the urgency of evidence-based outbreak management plans to predict, prevent or interrupt spreading of these arboviruses to non-endemic countries in temperate regions. We here present the results of three mark-release-recapture experiments conducted in a peri-urban area of North-East Italy to estimate the spatio-temporal dynamics of the dispersal of  Reliable estimates of active mosquito dispersal and flight ranges are thus very hard to obtain. This is even more challenging in the case of exophagic and exophilic species which are particularly difficult to recapture in the frame of Mark–Release–Recapture (MRR) studies1.Knowledge about the spatio-temporal dynamics of dispersal of adult mosquito vectors of diseases is instrumental for understanding mosquito-borne disease transmission dynamics and for effectively calibrating control intervention in order to prevent or interrupt pathogen transmission. Active dispersal of adult mosquitoes is triggered by the need to find mates, sugar sources, resting sites and, in the case of females, hosts for blood-meals and oviposition sites and is highly affected by intrinsic species-specific flight capability, as well as by ecological  and climatic  conditionsAedes albopictus, the Asian Tiger Mosquito, the species which in the last 30 years has invaded all continents (except Antarctica) thanks to the passive transportation of its eggs mostly inside used tires and lucky bamboos, and has stably colonized not only tropical, but also temperate regions due to the capacity to overcome the cold months in a state of embryonic hibernation2. These capacities have turned the species into one of the most successful invasive animal species worldwide3 and in a global public health threat due to its competence to transmit a large number of exotic arboviruses (such as Chikungunya (CHIKV), Dengue (DENV),4 and Zika5). This is already testified by Ae. albopictus central role in large CHIKV epidemics in Indian Ocean in 2006–076 and in the Caribbean and Americas since 20157 and in several cases of autochthonous transmission of CHIKV and DENV in temperate countries8.For the above reasons, little is known about the flight range of Ae. albopictus females looking for a host and blood-fed females looking for oviposition sites. In the former case, most marked host-seeking females were collected within 100 m from the release site , while investigation carried out in Texas, on host-seeking females emerged from larvae marked with stable isotopes, showed that approximately 79% of them were found within 250 m from their natal site and all of them remained within a 1 km distance12. However, females released in the forest were shown to flight up to 1 km to reach an urban area with high densities of possible hosts13. In the case of oviposition-associated dispersal, studies exploiting the release of Rb-labelled females and recapture of Rb-marked eggs reported presence of Rb-marked eggs in ovitraps throughout the whole 160 m radius urban area sampled in Singapore14, while in Brazil most Rb-marked eggs and fluorescent dusted females were found within 100–200 m from a release site15. In Florida, Davis et al.16 showed that self-marked gravid Ae. albopictus female dispersal increased with time, but seemed to stabilize around 90 m from the initial marking site.Few MRR studies have been carried out to investigate the capacity to disperse of both host-seeking gravid Ae. albopictus females released after blood-meal and recaptured by sticky traps (STs), and obtained flight ranges of 90% of the released females ranging between 168 and 236 m17. The first aim of the present study - carried out with the same design, but in a peri-urban area in north-east Italy - was to understand whether the flight ranges assessed in Rome could represent a common feature of Ae. albopictus female dispersal associated to search of an oviposition site in temperate regions. In addition, we exploited the data to model the temporal dynamics of the dispersal. This parameter has been largely neglected in previous studies, despite its high relevance for models estimating the dynamics of spread of autochthonous transmission of exotic arboviruses in temperate regions, as well as for the calibration of the spatial/temporal scale of insecticide treatments to be implemented to prevent or interrupt spread of arbovirus outbreaks. In fact, the recent cases of autochthonous CHIKV and DENV transmission in Europe8 highlight the urgency and timeliness to improve preparedness to predict, prevent or interrupt spreading of these and other exotic tropical arboviruses to non-endemic northern countries.In 2008, we carried out 3 MRR experiments within a highly urbanised area in the city of Rome  to analyse dispersal of florescent dusted Ae. albopictus females were collected out of the 3,959 released ones , 14.4% males , while for the remaining 6.4%  was not possible to determine the gender, due to bad preservation of carcasses glued on the STs. Some specimens, mostly females, of Culex sp., Culiseta sp.. and Aedes caspius were also found in the STs but not counted.A total of 356 marked vs 500 m radius, 55 STs vs 96 STs) in Rome . This may be due to the larger sampling area and the higher number of STs used in the present study as opposed to Rome and/or to an inherent bias in using STs as recapture method . However, it is worth to note that the recapture rates obtained in both studies are generally higher than those obtained in MRR studies on Ae. albopictus females dispersal after blood-meal carried out with different recapture approaches13. These results confirm that STs are a valuable tool for MRR studies on Ae. albopictus ovipositing or resting females .The recapture rates at 11 days from release were 8.8% (MRR1), 12.9% (MRR2), 3.0% (MRR3) and significantly different among the three experiments  and 50% (FR50) of marked females in each MRR experiment. Cumulative MDTs were 110 m (days 2–11), 77 m (days 2–16) and 68 m (days 2–16) in MRR1, MRR2 and MRR3, respectively. Daily MDTs were statistically different between the first two MRRs and MRR3 , but not between MRR1 and MRR2 . The maxODTs were 463 m (day-4), 433 (day-5) and 375 (day-6) in MRR1, MRR2 and MRR3, respectively. The flight ranges were always ≤50 m for FR50 and ≤252 m for FR90 (Table 10(annulus median distance + 1) and release was significant as shown by a Likelihood ratio test P < 0.01, so the three lines were not parallel between them: MRR1 vs MRR2: t = 4.98, P < 0.01; MRR1 vs MRR3: t = 4.08, P < 0.01; MRR2 vs MRR3: t = −9.06, P < 0.01).Table 90. The 90% percentile values reached about 250 m after 2 (MRR1) and 14 (MRR2) days since release (this estimate was not reached during the sampling period in MRR3). On average, the 90% percentile of the distance up to which mosquitoes are expected to fly was stable in MRR1, but it was predicted to increase with time in MRR2 (from <150 m at day-2 to ~250 m at day-14) and in MRR3 (from <100 m at day-2 to ~200 m at day-14), although with shorter estimates and wider confidence intervals due to the lower numbers of mosquito recaptured  was estimated for MRR3 Fig. .Ae. albopictus females kept under semi-field conditions for the duration of the experiments was shown not to be affected by the fluorescent dyes. In fact, no differences in survival between marked  and unmarked  females were recorded in MRR1, MRR2 and MRR3, respectively .The survival of marked Ae. albopictus females and the oviposition pattern of blood-fed females simultaneously to the released ones and kept under semi-natural conditions for the entire length of the experiments  between the frequency of recaptured nts Fig. . No corrnts Fig. , the oviAe. albopictus females in a peri-urban habitats in order to confirm or dispute that evidence obtained in a similar study carried out in a highly urbanized study area in Rome17 can be generalised and represent a reliable estimate of Ae. albopictus female dispersal associated to search of an oviposition site in other temperate regions.The first aim of the present work was to study active dispersal of blood-fed Ae. albopictus blood-fed females were recaptured within 250 m from the release site (FR90 = 177–252 m), a range consistent with estimates from the results obtained in Rome (FR90 = 168–23617). Moreover, the comparison between the oviposition patterns of females kept under semi-field conditions and the recapture rates in MRR2 supports the hypothesis that the observed dispersal was mostly triggered by the need of Ae. albopictus females released at the blood-fed stage to find a suitable oviposition site after blood-meal digestion  or the size (500 m vs 250 m radius) of the two study areas tested. However, it is important to highlight that the results obtained are based on mosquitoes reared under optimal conditions, while adult fitness and behaviour in nature are certainly affected by several features such as competition for trophic resources at the larval stage and climatic conditions. For instance, Mori18 reported that Ae. albopictus emerging from larvae reared under crowded conditions dispersed further than those reared in uncrowded conditions. Also, increased skip oviposition was reported in adults exposed to low-quality/crowded versus high-quality/uncrowded larval habitats16. These observations imply that gravid females will move greater distances when larval habitat quality decreases and competition for resources increases. On the other hand, extreme climatic conditions negatively affect survival and dispersal. In fact, compared to results from MRR1 and MRR2 , results from MRR3  show a much lower recapture rate, a delayed oviposition dynamics between released females and females kept in sheltered cages, and lower-distance dispersal.The results obtained showed that 90% of marked ion Fig. . TherefoAe. albopictus females increased with time, but seemed to stabilize around 90 m from the initial marking site16. Our analyses of the temporal patterns of dispersal revealed that the distance travelled by blood-fed females is longer and does not always increase over time. In fact, results from MRR1 showed that the potential exists for a very rapid dispersal, as it is predicted that about 90% of marked mosquitoes have flown within 250 m in 72 hours since release and the remaining 10% have flown even longer .The obtained results are very relevant for the planning of control activities to prevent autochthonous transmission of exotic arboviruses in temperate areas, an occurrence which is likely to become recurrent, as testified by the Chikungunya outbreak occurred in central Italy in summer 2017 with almost 500 human infected cases26. Overall, this reinforce the claim that control activities carried out around the place of residence of infected cases are not sufficient to control an emerging arbovirus outbreak, not only due to the importance of outdoor activities in the spread of Aedes-borne diseases in temperate countries27, but also due to operational constraints in intervening in an adequate area in due time.In conclusion, the above results suggest that insecticide treatments aimed to prevent risk of autochthonous outbreaks of exotic arboviruses in temperate regions may fail to reach a non-neglectable part of the potential vector population even if implemented in a 200-m buffer after reports of an infected case to health authorities, considering an average of 6 days delay between symptom onset and detectionThe study area (≈80 hectares) lies within the municipality of Piove di Sacco  and is characterized by either peri-urban or rural habitats Fig. . The perrd (MRR1) and 24th (MRR2), and in September 9th (MRR3) 2009, with the agreement of Piove di Sacco municipality. Temperature data during the experiments  at larval density ∼1 larva/mL with cat pellets as food (Friskies® Adults). Pupae were transferred to 40 cm cubic cages, where adults emerged and were maintained with 10% sugar solution until the day of release. Eggs, larvae and adults were always kept outdoors in a site sheltered from direct sunlight and wind.28. Blood-fed females were transferred into paper-cups closed by a net in groups of 20, and marked by gently dropping orange fluorescent dust  into the cup. Marked females were then transferred back into the cages and provided with a 5% sugar solution. In the evening, cages were brought by car to the release site in the centre of the study area inside paper boxes. At 10 PM - when Ae. albopictus is known to be inactive2 - marked females were released by opening the cages, which were left open all night long, in order to leave time to mosquitoes to fly away. Alive and dead individuals found in the cages the following morning were counted and excluded from the total number of released mosquitoes.In the morning of each release, 2–4 days-old females were fed with defibrinated fresh bovine blood by a hand-made membrane feeding system29 located at ground level in sheltered positions and geo-referenced, using a global positioning system device (Garmin GPSMAP 60CSx). In order to have the same collection-power at every distance from the release site, the study area was virtually subdivided into 10 concentric annuli (from 50 to 500 m radius) around the release site, with the number of STs/annulus increased from the centre to extremes of the sampling area to maintain the proportion ST/area to an average density of ≈1 ST/8,200 m2 in each annulus lus Fig. . All STs30.Recapture rates were calculated for each MRR experiment as the proportion of the number of recaptured marked mosquitoes over the total number of released ones. Recapture rates in the three MRRs experiments were compared by a Bernoulli GLM, with the number of mosquitoes recaptured within the first 11 days as response variable and the MRR experiment as covariate. These and the following statistical analyses were carried out using the statistical software Ret al.17, i.e. estimating: (i) the Mean Distance Travelled during the entire sampling period ; (ii) the daily MDTs for the first 6 days after release; (iii) the maximum Observed Distance Travelled (max ODT), corresponding to the linear distance from the release site to the farthest positive ST ; and iv) the Flight Ranges (FRs) based on the linear regression of the cumulative number of expected recaptures (ERs) from each annulus (x-axis) on the log10 (annulus median distance +1). FR50 and FR90 values were calculated from the equation of regression line as 50% and 90% of the largest ER value, respectively33. Additionally, the linear regression of the cumulative number of expected recaptures (ERs) was also computed considering the log10 (annulus median distance +1), the release and their interaction to test the difference between flight ranges in each release (by comparing the slopes of the three regression lines).To allow direct comparison of the results obtained in the present study and those previously obtained in Rome, dispersal of the released mosquitoes was analysed as in Marini Ae. albopictus females are expected to be found conditional to the time of release, dispersal data were further analysed by means of the following three models: (1) a GLM with Bernoulli distribution and logit link to model the probability to detect a marked mosquito conditionally to days and distance from release, and therefore to estimate the probability to detect a marked mosquito at a given distance. The response variable in the model is the presence/absence of marked mosquitoes into the STs, while covariates are the distances of STs from the release site and the days after release; (2) a GLM with Gamma distribution and log link (hereafter Gamma GLM) to model the distance travelled by marked mosquitoes conditional to the days after release and to compute the percentile of the distance travelled by recaptured marked mosquitoes. The response variable in this model is the distance from the release site at which marked mosquitoes were captured; in compliance with the Gamma distribution constrain, only strictly positive distances are considered. Covariate are the days after release; (3) a Zero Altered Gamma model (hereafter ZAG34), which accounts for the recaptured mosquitoes into the ST located on the release site (zero distance) and allows to estimate the percentile of the distance at which mosquitoes are expected to be found, weighted for the probability that marked mosquitoes collected actually moved from the release site. Therefore, in this model the distance travelled is modelled conditional to the days after release by means of a two-step model. First, a Bernoulli model with a binary response variable  is applied to evaluate the probability that a marked mosquito moves away from the release site. Second, a Gamma GLM is fitted on distances from the release site at which marked mosquitoes were captured. Covariate was the days after release for both sub-models. In all the three models, confidence intervals for the estimated quantities are computed by non-parametric bootstrap applied to original data.To provide estimates of the distance up to which marked Ae. albopictus blood-fed females were removed from the cages by manual aspiration before  and after  the marking procedure described above. Both groups were kept in 25 cm cubic cages kept outdoors in sites sheltered from direct sunlight and wind and provided with water bowl for egg-laying and 5% sugar solution ad libitum.Before each MRR experiment, Number of dead mosquitoes was recorded daily for the entire duration of the MRR experiment. Mortality rates in the two groups were analysed using the Kaplan-Meier method and comparing survival curves by Log-rank test.During MRR2 and MRR3, individual marked blood-fed females  reared and handled as described above, were kept in plastic cups closed with mosquito net and lined with filter paper, kept in sheltered sites outdoors. Oviposition activity was monitored daily for the entire length of the MRR experiments, by counting the number of eggs deposited on the filter paper in each cup. The correlation between the number of marked mosquito females collected in MRR2 and MRR3 and the number of the ovipositing females during the same intervals was assessed by Pearson’s correlation.Supplementary Information


This deviation in scores between the two tests was greater as z scores decreased for both tests , with no such relationship observed for age-equivalents. These results indicate the conversion of raw scores between the KBIT-2 verbal scale and the BPVS3 may be a more valid method for the comparison or combination of study samples with DS compared to the use of standardised scores. Such comparisons or combinations will aid our understanding of cognitive variations and factors associated with these variations within the population with DS.Down syndrome (DS) is the most common genetic cause of intellectual disability. There is, however, considerable variation in cognitive abilities between those with DS, with some individuals scoring at floor on some tests, particularly for age-standardised outcomes. This variation and these floor effects can pose a problem for comparing and combining study populations when different standardised measures have been used to assess individuals’ cognitive abilities, for example combining results across studies to investigate genetic or other factors associated with cognitive abilities. To facilitate this comparison and combination of study populations assessed using different tests of verbal abilities, we administered two commonly used standardised tests of receptive language, the Kaufmann Brief Intelligence Test 2 (KBIT-2) verbal scale and the British Picture Vocabulary Scale 3 (BPVS3) to 34 adults with DS (age range 19–59) to investigate relationships between outcomes for these two tests. We found a very strong correlation between raw scores for the KBIT-2 verbal scale and the BPVS3, and determined equations to convert between scores for the two tests. Intraclass correlations between the two scales for age-equivalents and calculated  Down syndrome (DS), caused by the triplication of chromosome 21, is the most common genetic cause of intellectual disability (ID), and has a UK live birth incidence of approximately one in 1000 . DespiteTo facilitate the comparison and combination of datasets from studies that have used different cognitive outcome measures it is vital to understand how scores on different tests compare. Ideal measures for comparison or combination will be from commonly used tests, with tests of IQ being one such likely candidate as these are typically included in studies as a measure of general cognitive abilities. Several IQ tests have previously been used in studies assessing cognitive abilities in adults with DS, including tests of both verbal and non-verbal abilities [see Hamburg et al. (unpublished) for a review]. The standardisation of IQ tests typically allows for combination of age-adjusted IQ scores between studies in individuals who do not have an ID, however, in studies of adults with DS high floor effects are observed when converting to standardised IQ scores . This isAge-equivalent scores may provide an alternative to standardised IQ scores, although floor effects are still possible, albeit to a smaller extent than for standardised IQ scores. Further issues with age-equivalent scores come from their interpretation. Firstly, these scores describe the age at which a particular raw score is average, and do not account for the variation in raw scores at that age. Secondly, interpreting the difference between two age-equivalent scores depends upon the scores themselves as development is not linear; for example differences of 6 months for ages 5 and 5.5 years and for ages 12 and 12.5 years are not equivalent . In addiz scores relative to population norms  between the tests, to investigate the potential use of standardised measures to combine results from these two tests.Unlike IQ scores and age-equivalents, however, there is no standardisation for raw scores to allow for combination between different tests. To combine study results it is therefore essential to establish the relationship between raw scores for different tests. To aid in this, we administered two commonly used standardised tests of receptive language (the Kaufmann Brief Intelligence Test 2 (KBIT-2) verbal scale and the British Picture Vocabulary Scale 3 (BPVS3)) to a sample of individuals with DS to explore the relationship between scores for these tests. These tests were selected based on their easy and relatively quick administration requiring minimal verbal responses and previous use in a range of adults with DS, with test performance being considered a proxy for general cognitive abilities . We alsoWe recruited 34 adults aged 18+ years with DS  from the LonDownS Consortium adult participant pool . ParticiEthical approval was obtained for the LonDownS study from the North West Wales Research Ethics Committee (13/WA/0194). Where individuals had capacity to consent for themselves we obtained written informed consent in accordance with the Declaration of Helsinki. Where individuals did not have capacity to consent for themselves, a consultee was appointed and asked to sign a form to indicate their decision regarding the individuals’ inclusion based on their knowledge of the individual and his/her wishes, in accordance with the UK Mental Capacity Act 2005.Participants were administered the KBIT-2  and the The KBIT-2 was designed to measure verbal and non-verbal intelligence of individuals aged 4–90 years, and consists of a verbal and non-verbal scale, with the verbal scale containing two subtests, assessing verbal knowledge and riddles. For the verbal knowledge subtest then participants are given a word and asked to select the correct picture corresponding to the word from six options, and for the riddles subtest then participants are asked to identify the item described in a short riddle. Each subtest was started at item 1 and stopped after four consecutive incorrect answers.The BPVS3 was designed to measure receptive vocabulary for Standard English, particularly for individuals aged 3–16 years, but also being suitable for adults, and contains a verbal knowledge test. In this test participants are given a word and asked to select the correct picture corresponding to the word from four options. The starting point was chosen based on researchers’ prior knowledge of the individuals’ verbal ability; all individuals had participated in our previous studies , which iStarting points for test administration for the KBIT-2 and BPVS3 were therefore adapted as appropriate from the manuals to be suitable for a population with ID; for both tests the manuals provide different starting points based on individuals’ chronological age.z scores relative to population norms. z scores are a suitable alternative to standardised scores in those with an ID to determine deviations from typical age-expected performance across ages and test domains, due to the high floor effects when converting raw scores to standardised scores. z scores were calculated using the means and standard deviations in provided tables using the below formula:All analysis was performed using SPSS version 22. Raw scores were first determined for the KBIT-2 verbal scale and BPVS3. Using standardised tables provided in the test manuals, scores were converted to age-equivalents, and to i is in age group j. Using this formula, a z score of 0 represents typical age-expected performance, while a z score of –1 represents performance one standard deviation below this. For the BPVS3 the oldest age group with available means and standard deviations was 16 years 9 months to 16 years 11 months, and so the mean and standard deviation for this age group was used to determine z scores for all individuals as individuals in our sample were aged 19 years and above. For the KBIT-2 verbal scale, we therefore used the mean and standard deviation for a comparable age group to determine z scores for all individuals . The BPVS3 does not provide standardised IQ scores below 70, and so no comparisons were made for standardised IQ scores between the KBIT-2 verbal scale and BPVS3.where individual t-tests. Intraclass correlational analysis assesses the reliability of scores by assessing absolute agreement, accounting for their absolute values in addition to the relationship between them.Firstly, we performed Pearson’s correlational analysis to determine the relationship between raw KBIT-2 verbal scores and BPVS3 scores. Based on this relationship we then determined linear regression equations to convert between test scores. Using these equations we calculated predicted KBIT-2 verbal scores and BPVS3 scores based on actual raw scores from the other test, and compared the absolute values of raw and predicted scores within each test using (a) intraclass correlational analysis and (b) paired samples t-tests to compare absolute values of age-equivalents and z scores between KBIT-2 verbal scores and BPVS3 scores. Based on these results, we also performed Pearson’s correlational analysis for age-equivalents and z scores to determine whether deviation in scores between the two tests was associated with absolute scores on either test.Secondly, we performed (a) intraclass correlational analysis and (b) paired samples z scores relative to population norms for the KBIT-2 verbal scale and BPVS3 can be seen in Table z scores for KBIT-2 verbal scores and BPVS3 scores can be seen in Figure n = 27 and n = 15 at floor, respectively).Information on raw scores, predicted scores, age-equivalents, and r = 0.96, p > 0.001, see Figure Correlational analysis indicated a very strong correlation between raw scores for the KBIT-2 verbal scale and the BPVS3 (p < 0.001) with no significant difference between the two scores . Similarly, comparing raw and predicted BPVS3 scores indicated a high intraclass correlation coefficient between the scores , p < 0.001) with no significant difference between the two scores .Using these formulae we calculated predicted BPVS3 scores based on actual raw KBIT-2 verbal scores, and predicted KBIT-2 verbal scores based on actual raw BPVS3 scores  and 0.65 , p < 0.001), respectively.Intraclass correlation coefficients for age-equivalents Figure  and z scs Figure  between z scores were significantly higher for KBIT-2 verbal scores compared to BPVS3 scores .Both age-equivalents and z scores between KBIT-2 verbal scores and BPVS3 scores, we finally determined whether there were any relationships between the size of these differences and the values of age-equivalents and z scores respectively for the KBIT-2 verbal scale and BPVS3, i.e., whether the degree of deviation in scores from the two tests was associated with scores on either test.Based on these results, which indicated differences in absolute values of age-equivalents and r = 0.33, p = 0.083) or the BPVS3   and the BPVS3  , while no such relationship was observed for age-equivalents.These results indicated there was a greater deviation in We present results comparing scores from two commonly used tests to assess receptive verbal abilities in individuals with DS, the KBIT-2 verbal scale and the BPVS3, with scores on these tests considered as a proxy for general cognitive abilities. Raw scores showed a very strong correlation between tests, and we determined equations for converting scores between the tests. Using these equations to predict test scores, we demonstrated very high intraclass correlation coefficients with very small mean differences between actual raw scores and predicted scores for both the KBIT-2 verbal scale and the BPVS3, indicating the high reliability of the equations. These equations therefore allow for the reliable conversion of scores between the KBIT-2 verbal scale and the BPVS3, with the very strong correlation between scores for these two tests indicating they assess the same underlying construct(s). This conversion will allow for the comparison or combination of data from studies using these two tests, with the latter allowing for better-powered studies requiring large sample sizes, such as those investigating genetic associations.z scores relative to population norms for the KBIT-2 verbal scale and the BPVS3. Intraclass correlation coefficients between both age-equivalents and z scores for the two tests were high. However, scores for both outcomes were significantly higher for the KBIT-2 verbal scale compared to the BPVS3, with the mean values of these scores suggesting the BPVS3 to give an age-equivalent of approximately 6 months younger and a z score one and a half standard deviations further from typical performance compared to the KBIT-2 verbal scale. This indicates a systematic difference in these standardised scores, suggesting it may be unreliable to compare or combine them. Further, this deviation in z scores between the two tests was greater for those with poorer abilities, indicating the comparison of z scores from the two tests becomes more unreliable as abilities decrease, with no such relationship observed for age-equivalents. Though analysis for age-equivalents did not include those with an age-equivalent at floor or ceiling, excluding these individuals from analysis for z scores did not change the patterns of results observed (results not presented). Based on our results we are unable to determine whether the BPVS3 is underestimating scores or the KBIT-2 verbal scale is overestimating these, or a combination of the two. In addition, for the calculated z scores, this difference may suggest that the BPVS3 has a lower floor compared to the KBIT-2 verbal scale.In addition, we compared age-equivalents and calculated z scores are not suitable for use to compare or combine study samples and results using these two tests. As previously reported, high floor effects in age-adjusted standardised IQ scores for individuals with an ID means these scores are not sensitive to individual differences for those at floor, and so are also often not suitable for use  are often useful for tracking change within individuals with ID.Our finding of higher age-equivalents and In addition to understanding how scores on different tests relate to each other being essential to compare or combine datasets, it is important to understand these relationships when IQ tests may be used to assess individuals to determine their needs and appropriate support. z scores, with population data obtained from the KBIT-2 and BPVS3 manuals. Firstly, the population data available was not collected from individuals with DS, which may limit its generalizability for converted age-equivalents and z scores in the present study. Secondly, differences within the populations used to obtain population data may impact upon its utility; KBIT-2 population data was obtained from individuals in the United States, and BPVS3 population data was obtained from individuals in the United Kingdom. Finally, based on the availability of population data from the BPVS3 we used the population mean and standard deviation for individuals aged 16 years 9 months to 16 years 11 months to calculate z scores (the oldest available age group). Although the BPVS3 is suitable for use in adults, it is typically used in individuals aged 16 years and under, and norms are only available for individuals aged 3 to 16 years. To improve comparisons we used population data from a similar age group to calculate z scores for the KBIT-2 verbal scale . The use of population data from these age groups rather than using population data for adults with a similar chronological age to participants may therefore have altered individuals’ “true” z scores relative to population norms. As our data analysis was comparing z scores for the two tests rather than interpreting z scores relative to population norms this does not affect our conclusions, but is an important consideration to note. Another limitation relates to our sample size; while this was sufficient to demonstrate a number of significant relationships in our data, a larger sample may aid in confirming possible relationships between the difference in age-equivalents for the two tests and age-equivalent scores using either the KBIT-2 verbal scale or the BPVS3.The main limitation of our study relates to the use of population data to determine age-equivalents and Within this study, participants demonstrated a range of scores on the KBIT-2 verbal scale and BPVS3, suggesting the results presented here should be relevant to a wide range of studies of individuals with DS. We recruited individuals with a wide age range, with non-trisomy 21 DS, and with psychiatric and neurological comorbidities to ensure results were generalizable to the wide spectrum of individuals with DS. As analysis compared results within individuals rather than between individuals such variations do not impact upon the reliability of our conclusions. Future studies should also include other populations with an ID or younger individuals with DS to determine whether these results may be applicable to these other populations, and also use other IQ tests  to determine relationships between scores on different tests. Understanding these relationships will facilitate the comparison of studies, and allow for the combination of data from different studies, an essential first step in understanding population variations and factors associated with these.The LonDownS Consortium principal investigators are Andre Strydom (chief investigator), Department of Forensic and Neurodevelopmental Sciences, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, United Kingdom, and Division of Psychiatry, University College London, London, United Kingdom; Elizabeth Fisher, Department of Neurodegenerative Disease, Institute of Neurology, University College London, London, United Kingdom; Dean Nizetic, Blizard Institute, Barts and the London School of Medicine, Queen Mary University of London, London, United Kingdom, and Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore; John Hardy, Reta Lila Weston Institute, Institute of Neurology, University College London, London, United Kingdom, and United Kingdom Dementia Research Institute at UCL, London, United Kingdom; Victor Tybulewicz, Francis Crick Institute, London, United Kingdom, and Department of Medicine, Imperial College London, London, United Kingdom; and Annette Karmiloff-Smith, Birkbeck University of London, London, United Kingdom (deceased).https://doi.org/10.6084/m9.figshare.6553514.v2.The dataset analysed for this study can be found in the figshare repository at AS conceived the adult cohort study in conjunction with LonDownS principal investigators. CS designed the study and data analysis, analysed the data, and drafted the initial version of the report. CS and SH performed data collection. All authors contributed to revision and editing of the report.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic β-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse KATP inhibitors are known to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of a mammalian KATP channel bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. We found all three drugs bind within a common pocket in SUR1. Further, we found the N-terminus of Kir6.2 inserted within the central cavity of the SUR1 ABC core, adjacent the drug binding pocket. The findings reveal a common mechanism by which diverse compounds stabilize the Kir6.2 N-terminus within SUR1’s ABC core, allowing it to act as a firm ‘handle’ for the assembly of metastable mutant SUR1-Kir6.2 complexes.ATP-sensitive potassium (K ATP) channels, wherein four SUR.x subunits form a complex with four subunits of an inwardly rectifying potassium channel, Kir6.1 or Kir6.2  (ATP-binding cassette transporters (ABC transporters) comprise a large protein superfamily responsible for transporting diverse molecules across cell membranes . Uniquelr Kir6.2 . KATP chthe cell . Expressthe cell . In pancecretion . Gain-ofsulinism . Among tinafter) .ATP channel inhibitors commonly used to treat Type 2 diabetes are efficient pharmacochaperones for correcting surface expression defects of certain trafficking-impaired mutant KATP channels, including sulfonylureas (SUs) and glinides , repaglinide , and CBZ, interact with the channel to affect channel biogenesis and function. Using single-particle cryo-electron microscopy (cryoEM), we determined structures of the channel in the presence of GBC, RPG, and CBZ, and without pharmacochaperone. The structures show that like GBC , RPG andATP bound to GBC and ATP (2+) but varying the pharmacochaperone by alternatively including 30 μM RPG (referred to as the RPG/ATP state), 10 µM CBZ (referred to as the CBZ/ATP state) or the drug vehicle 0.1% DMSO (referred to as the ATP-only state) in the sample. Further, as an additional control we determined the structure of channels without any pharmacochaperone or ATP (referred to as the apo state).For structure determination, a FLAG-tagged hamster SUR1 and a rat Kir6.2  were overexpressed in the insulinoma cell line INS-1, and the channel complex affinity purified via the FLAG-epitope tag as described for our recent cryoEM structure determination of K and ATP . These c and ATP . To allo and ATP , data weInitial data processing in RELION with C4 symmetry imposed yielded one dominant 3D class for each dataset, with an overall reported resolution ranging from ~4 Å for the RPG/ATP and CBZ/ATP states to ~7 Å for the ATP-only and the apo states . As with2+ (Creative Molecules Inc) (Purified Kles Inc) . CBDPS wles Inc)  using crles Inc)  partner ATP channel formed by a SUR1-Kir6.2 fusion protein bound to repaglinide ; Colin G. Nichols (Reviewer #3); Jeffrey Agar (Reviewer #4).Thank you for submitting your article "Mechanism of pharmacochaperoning in KThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:2+, which provides an excellent confirmation of earlier photoaffinity labeling studies.The reviewers appreciate the importance of the new cryogenic-EM structural data providing key evidence of the commonality of binding sites for different channel inhibitors and pharmacochaperones. This is also the first observation in cryo-EM of ATP binding to NBD1 of SUR1 in the absence of MgHowever, as the paper is focused on the hypothesis that the pharmacochaperone effect occurs through drug stabilization of the interaction between the Kir6.2 N-terminus (KNt) and the SUR ABC core domain, revisions are needed to address weaknesses in the evidence for this hypothesis.Essential revisions:1) Please make proper acknowledgment of the prior structural evidence, in the 2018 Wu et al. paper, for binding of the KNt to the SUR core domain. This paper is mentioned, en passant, as revealing a weak and disconnected cryoEM density that 'fueled speculation' that the KNt can be located within the central cavity of the SUR1 core. But this is a bit dismissive. The Wu et al. paper makes a rather strong case for this argument, revealing near contiguous density coming off the Kir6.2 subunit towards the cavity and with only a short gap to the density in the cavity, whereas the current manuscript shows what seems to be a more disembodied density in the cavity, and appears to be at no better resolution than that of Wu et al. Moreover, Wu et al. showed additional correlation in so far as there was no density in the SUR1-Kir6.2 fusion structure (in which the N-terminus is not free to act as an extended peptide).Please also comment on whether in the current structures there is any continuity between Kir6.2 and the density attributed to the N-terminus of Kir6.2 within the core of SUR1 , and what in the new structures makes it convincing that it is the N-terminus. The present functional data are not sufficient to make this conclusion.2) Address the weaknesses in the new experimental evidence for the binding of the KNt and its role in the pharmacochaperone effect:A) Potentially the strongest new evidence is the MS identification of cross-linked peptides including the KNt and the ABC core. However, there are major concerns that the XL-MS workflow used could produce false positives, particularly because of the absence of an alkylation step to reduce the chance of disulfide scrambling. These experiments should be repeated using an alkylation step and with tighter mass tolerances, using a well-established or validated XL-MS workflow.  Complete details of the experimental methods and the XL-MS raw data files should be included.It should also be acknowledged that the cross-linking is done on purified protein and not on channels in membranes, so it is possible the protein adopts conformations it might not normally do under physiological conditions.On this question of false positive interactions, note that some of the SUR1-SUR1 crosslinking residues seems very far apart in both propeller and quatrefoil structures. For instance, K915 to K1412 looks to be ~70Å yet the linker is only 15A.B) The electrophysiological tests of cross-linking of the Kir6.2-L2C mutant aim to provide functional evidence for KNt contacting the ABC core domain in an intact membrane under physiological conditions, however, in the opinion of the reviewers and the Reviewing Editor, these current results do not provide convincing support for the hypothesis. The text states that "co-expression of Kir6.2-L2C with WT SUR1 resulted in channels that displayed an accelerated current decrease in I2 (250µM) that was partially reversible by subsequent exposure to DTT[…]" In fact, both wild type channels and Kir6.2-L2C/SUR1-C1142A channels show a similar extent of inhibition during iodine application. If the difference between Kir6.2_L2C and controls is in the rate of current decrease, there is no quantification to support this claim. It is true that there was no DTT reversal apparent in the controls, but this reversal is only apparent in a subset  of the Kir6.2-L2C records. Were the experiments that showed more profound reversal only the ones with small currents ? Was the effect of DTT alone tested on wild-type or mutant channels? As for the drug block experiments, how are the effects of iodine differentiated from the effects of rundown ?It is unclear how best the authors can address these concerns and shortcomings in the physiological cross-linking experiments. Possibly more experiments along these same lines will make the effects more clear. Alternatively, given the apparent distance between the putatively cross-linked sites in the structure, it may be helpful to investigate the effects of a rapid, non-zero-length crosslinker such as a bis-MTS reagent. Although it would be preferable to include an improved version of these experiments, if this is not possible then these experiments should probably just be omitted from the revised manuscript.C) Although deletions at the N-terminus of Kir6.2 are said to abolish the pharmacochaperone effect (by interfering with the putative interaction of the KNt with the drug bound within the SUR core), there is a very clear pharmacochaperone effect on the ΔN5 mutation in Figure 4—figure supplement 1C (and data are not shown for the ΔN10 mutation). If indeed the KNt deletion prevents the interaction with the SUR-bound drug, it suggests that the pharmacochaperone effect may be independent of the KNt interaction. The weaker (but still not absent) pharmacochaperone effect with the addition of the F27S mutation (panel B) may simply indicate that the KNt deletion destabilizes the protein and the F27S destabilizes it further, with both effects independent of the drug binding. At a minimum, the results with ΔN10 (with no F27S) should be shown, and this alternative interpretation should be discussed.3) In Figure 3—figure supplement 1B,C, it is not possible to deduce if the mutations in SUR1 had any effect on drug block because it is very hard to distinguish current rundown from block in the records provided. The difficulty is even greater when the currents are very small as for the mutant channel. How was the% current measured in panel D? There really seems to be no steady state block by the drug  and there is no clear, rapid onset of block. The problem with these drugs is that they are largely irreversible and they block slowly at low concentrations. It would be better to test a single drug concentration (preferably 100nM) on each patch. Alternatively, the S1238Y mutation of SUR1 could be exploited for comparisons as sulfonylureas block these mutant channels reversibly and at lower affinity.4) The value of the Rb+ efflux experiments in Figure 3 is unclear and the conclusions drawn from these experiments may be overstated. The drugs were applied for up to 40 min. Is this long enough to affect surface trafficking of SUR1? Can it be concluded if the data primarily represent the effect of the drug on channel inhibition? The authors argue that differences between mutational effects on the ability of repaglinide and carbamazepine to inhibit flux confirm that the mutations are directly in the drug binding site and do not have an indirect-allosteric effect. At first glance, the effects of mutating these residues in the binding pocket on the ability of the two drugs appear to be similar . Is flux a sensitive enough measurement to make such distinctions? Why were mutations tested using Rb+ efflux, rather than patch clamp? Efflux is both determined by the number of channels in the membrane and by the ability of the drug to block the channel. So they can't state 'mutations reduced the sensitivity of the channel to inhibition' – unless they have shown that changes in surface expression do not occur on the time scale of their experiments.Reviewer #1:In this manuscript, Dr. Shyng and colleagues presented cryo-EM structures of KATP channels bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. All three compounds are clearly resolved in these structures and showed that chemically diverse KATP channel inhibitors interact with the channel through the same binding pocket. In addition, this study provided structural information on how N-terminus of the channel interacts with the transporter and stay in the closed conformation.Manuscript is very well written, a lot of structural and functional data presented to confirm conclusions drawn from the structural work.eLife.Overall, I do not have cryo-EM specific experimental concerns and consider this manuscript be ready for publication in Reviewer #2:The authors have provided five cryo-EM structures of the KATP channel complex in association with ATP and either glibenclamide , repaglinide (RPG), carbamazepine (CBZ), or no drug, and in the absence of both drug and ATP. The data show that all 3 drugs bind in the same pocket on SUR1. They also identify some density near the drug binding site in the ABC core of SUR1 that they attribute to the unresolved N-terminus of Kir6.2 . The structural data are complemented by data showing the effect of mutations in the drug binding pocket on the ability of drugs to chaperone SUR1 to the plasma membrane (as indicated by complex glycosylation of SUR1).In addition, the authors provide data showing the effect of drug binding site mutations on Rb+ efflux (which reflects KATP channel activity in the whole cell) and excised patch recordings of the effects of GBC and RPG on KATP currents in WT and W1297 mutant channels. The location of the N terminus within the SUR1 cavity is explored in two ways: by chemical crosslinking followed by mass spectrometry, and by examining the ability to crosslink residues in SUR1 and Kir6.2 by engineered cysteine pairs.2+, which provides an excellent confirmation of earlier photoaffinity labeling studies. The surface expression data is also interesting and supports the idea that the drugs chaperone SUR1 to the plasma membrane by binding within the same drug binding pocket as that which is linked to channel inhibition. Surprisingly, since the lab has an excellent reputation for KATP channel electrophysiology, the functional data presented in this paper is weak and it is difficult to draw strong conclusions as to the mutational effects on drug binding or on the cross-linking of the N-terminus of Kir6.2 to SUR1 from the data as presented.The cryo-EM structures in complex with RPG and CBZ are novel, and should be published. This is also the first observation in cryo-EM of ATP binding to NBD1 of SUR1 in the absence of MgFigure 3—figure supplement 1B,C. It is not possible to deduce if the mutations in SUR1 had any effect on drug block because it is very hard to distinguish current rundown from block in the records provided. The difficulty is even greater when the currents are very small as for the mutant channel. How did they measure the% current in panel D – there really seems to be no steady state block by the drug  and there is no clear, rapid onset of block. The problem with these drugs is that they are largely irreversible and they block slowly at low concentrations. It would have been better to have tested a single drug concentration (preferably 100nM) on each patch. Alternatively, the S1238Y mutation of SUR1 could be exploited for comparisons as sulfonylureas block these mutant channels reversibly and at lower affinity.The authors provide persuasive structural evidence as to which residues line the drug-binding pocket. As such, the value of the Rb+ efflux experiments in Figure 3 is unclear and the conclusions drawn from these experiments may be overstated. The drugs were applied for up to 40 min. Is this long enough to affect surface trafficking of SUR1? Can it be concluded if the data primarily represent the effect of the drug on channel inhibition? The authors argue that differences between mutational effects on the ability of repaglinide and carbamazepine to inhibit flux confirm that the mutations are directly in the drug binding site and do not have an indirect-allosteric effect. At first glance, the effects of mutating these residues in the binding pocket on the ability of the two drugs appear to be similar . Is flux a sensitive enough measurement to make such distinctions? Why were mutations tested using Rb+ efflux, rather than patch clamp? Efflux is both determined by the number of channels in the membrane and by the ability of the drug to block the channel. So they can't state 'mutations reduced the sensitivity of the channel to inhibition' – unless they have shown that changes in surface expression do not occur on the time scale of their experiments.Figure 5. Most of their functional evidence for the 6.2 N-terminus contacting the ABC core domain comes from this figure. The authors state that "co-expression of Kir6.2-L2C with WT SUR1 resulted in channels that displayed an accelerated current decrease in I2 (250µM) that was partially reversible by subsequent exposure to DTT[…]" In fact, both wild type channels and Kir6.2-L2C/SUR1-C1142A channels show a similar extent of inhibition during I2 application. If the difference between Kir6.2_L2C and controls is in the rate of current decrease, there is no quantification to support this claim. It is true that the authors observe no DTT reversal in their controls, but this reversal is only apparent in a subset  of their Kir6.2-L2C records. Were the experiments that showed more profound reversal only the ones with small currents ? Did the authors test the effect of DTT alone on wild-type or mutant channels? As for the drug block experiments, how are the authors differentiating the effects of iodine from the effects of rundown ?Reviewer #3:This is an interesting paper, in a series with the earlier reports of Martin et al. on KATP channel structure, that provides key evidence of the commonality of binding sites for different channel blockers, and intriguing mechanistic connection between the SUR1 subunit and the Kir6.2 subunit. There are a few points that should be addressed:1) The paper is generally very well written, but should be carefully checked for minor typos and use of commas. One point of contention: 'evidence' is for or against an argument, 'evidence' does not 'establish' anything scientifically. In a couple of places, 'evidence' should be replaced by 'data' or perhaps 'results'.2) The authors mention the 2018 Wu et al. paper, en passant, as revealing a weak and disconnected cryoEM density that 'fueled speculation' that the KNt can be located within the central cavity of the SUR1 core. But this is a bit dismissive. The Wu et al. paper makes a rather strong case for this argument, revealing near contiguous density coming off the Kir6.2 subunit towards the cavity and with only a short gap to the density in the cavity, whereas the current manuscript shows what seems to be a more disembodied density in the cavity, and appears to be at no better resolution than that of Wu et al. Moreover, Wu et al. showed additional correlation in so far as there was no density in the SUR1-Kitr6.2 fusion structure (in which the N-terminus is not free to act as an extended peptide). This is not to detract from the authors' conclusions here, but more credit should be given to the previous paper.3) The major potentially definitive evidence in the current paper for the disposition of KNt is the mass spec data. In this regard, it is common to designate a fragmentation pattern as observed in the peptide fragmentation spectrum from tandem MS on the corresponding peptide sequence. Addition of the fragmentation information would make it easier to interpret the data.4) There are a couple of assertions of likelihood in the Discussion for which the evidence seems a bit thin. In particular, why is it the case that by trapping KNt in the central cavity, SUR1 is 'likely' to prevent a PIP2-bound conformation in Kir6.2? The PIP2 binding site is not in the distal N terminus.Reviewer #4:This review concerns only the cross-linking MS (XLMS) results. A summary of my review is that the XLMS workflow is not a "typical" workflow, has at least one fundamental flaw and lacks sensitivity, and the cross-linking results could well be false positive.XLMS is the most technically demanding and specialized type of biological MS experiment. As a result, expert users tend to rely upon well-established XLMS workflows that minimize false-positive IDs. It appears, however, that the authors have created a novel XLMS workflow , and combined this with novel methods for sample preparation . While the individual components of their workflow  are reasonable, have they been proven to work well (in combination) during XL-MS?This reviewer's recommended "fix" is to employ a start-to-finish XL-MS workflow  from a well-established group. The leaders in this field are Albert Heck (https://experiments.springernature.com/articles/10.1038/nmeth.3603) and Lan Huang . Note that all expert groups employ specialized, fit-for-purpose data analysis and validation methods . If the authors (and editor) choose the risky approach of using the author's current workflow, I include suggestions below for some de-risking.Five major concerns of the current XL-MS methods include: 1) The authors do not appear to employ a well-established XL-MS workflow (or do not reference one). The potential for false positives (via S-S scrambling) increases, the sensitivity of their analysis suffers, and alternative results (i.e. cross-links that don't fit the proposed model) will not be detected. 2) The cross-linking reaction is not described in sufficient detail to repeat or to critique and a reference to the cross-linking method does not appear to have been included. 3) The omission of the reduction step  is necessary with this cross-linker and was appropriate. However, the omission of the alkylation step will result in "free" cysteine containing peptides, which will certainly exchange (via thiolate-disulfide interchange) with the DSP S-S bond. This study must be performed with a denaturing alkylation step immediately following cross-linking and quench (prior to digestion). 4) No proper cross-link validation step was included. 5) The engineered disulfides should be detected directly by XL-MS analysis .ATP channel revealed by cryo-EM" for consideration by eLife. Your article has been re-reviewed by four peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Vera Y Moiseenkova-Bell (Reviewer #1); Colin G. Nichols (Reviewer #3); Jeffrey Agar (Reviewer #4); Michael Puljung (Reviewer #5).Thank you for resubmitting your work entitled "Mechanism of pharmacochaperoning in a mammalian KeLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in We appreciate the valuable new data in the paper, but our expert mass spectrometry reviewer still has serious concerns that the MS crosslinking results are too prone to false positives, without alkylation to prevent disulfide scrambling and a higher m/z resolution analysis of the crosslinking products. Given this concern combined with the weaknesses already pointed out in the functional disulfide crosslinking work, it seems that the central contention of the paper about the interaction between the KNt and the SUR1 core is not adequately supported by the experimental results.We understand that you may disagree with these concerns, but we consider them too important to overlook. At this point, we are rejecting the submitted manuscript, but we would welcome a new submission of a manuscript that addressed these concerns, most likely with new experimental results. Alternatively, if you think that we have just got it wrong, our rejection of the paper frees you to submit the paper to other journals.Reviewers #1 and #3 were satisfied with the revisions.Reviewer #4:The authors did formulate a general response to XL-MS concerns, and this stated (more or less) that the authors did not share my concerns  and therefore did not perform additional experiments. I would not myself consider publishing biologically relevant findings using a new method of analysis that hasn't undergone proper validation experiments, or an MS/MS accuracy cutoff of 1 Da, or not validating any putative cross-links with higher accuracy follow-up scans. However, I will admit to being on the more conservative side in such matters.My serious concern about false positives arises from free thiols that are part of the protein . There is a vast literature that not only predicts that thiolates (i.e. free cys on digested peptides) will attack the disulfide bond of DSP, displacing one of the two peptides, but that can even predict the rate constants of this reaction . In my own experience, if you have free thiols (on peptides) in the presence of disulfide bonds (on peptides), there will be complete scrambling of disulfide bonds. Here's the most worrisome part- following attack on the DSP disulfide bond by a Cys residue (from another peptide), the leaving group has Lys-conjugated DSP fragment that terminates in a thiolate. This can then attack another DSP cross-linked peptide and form a completely spurious crosslink .This is a fatal design flaw and is why I suggested they alkylate prior to digestion (which they didn't do). This problem of scrambling is more important than any concern about "overalkylation".Here is a link to a review describing the exchange rates: https://gmwgroup.harvard.edu/files/gmwgroup/files/369.pdfAnd here are a few published descriptions of disulfide scrambling in practice:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082428/https://www.ncbi.nlm.nih.gov/pubmed/17609855https://www.ncbi.nlm.nih.gov/pubmed/12441146https://www.ncbi.nlm.nih.gov/pubmed/10972999https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143640/If we assumed for the sake of argument that there were no problems with scrambling, the 1Da MS/MS cutoff is another fatal flaw (irrespective of other aspects of the XLMS experiment).The authors also use DSP in a way that it has never been used (I asked them to include a reference to their workflow and they didn't). DSP is usually chemically reduced before MS experiments (and compared to a non-reduced experiment). However, these authors extended Huang's concept of the "MS-cleavable" crosslinker from Collisional activation (CID) acting upon a sulfoxide bond , to ETD acting upon disulfides . Prior to using such a workflow to reach biologically significant decisions, there are a number of hoops authors jump through, one being a methods development study where they show a histogram of the length of the ID'd crosslinks (where the 70Å cross-link they ID'd would have been a problem). Here I invited them to prove their workflow on their engineered disulfide bond peptides, which like all other of my requests, they declined.And the authors don't really detect half-DSP conjugated lys residues- they detect peptides that happen to have modifications that sum to 88 +/- 1 Da (and there are a lot of ways to get to that mass).They use the insoluble crosslinker, rather than water soluble analogue.Unlike the studies of established labs, they don't bother using isotope labeled DSP to validate their results. These are commercially available.Most of these concerns are in my previous review.Reviewer #5:I find the new version of the Martin et al. manuscript to be greatly improved over the original. The inclusion of new data  was helpful as was their expanded discussion, particularly in reference to the Wu et al. paper. I am still not completely persuaded by their electrophysiology data in Figure 5 (I2 cross linking), but appreciate the changes they made in the main text regarding this figure, which is a much better description of the data. My remaining concerns are minor and can be addressed as changes to the Discussion. Essential revisions:1) Please make proper acknowledgment of the prior structural evidence, in the 2018 Wu et al. paper, for binding of the KNt to the SUR core domain. This paper is mentioned, en passant, as revealing a weak and disconnected cryoEM density that 'fueled speculation' that the KNt can be located within the central cavity of the SUR1 core. But this is a bit dismissive. The Wu et al. paper makes a rather strong case for this argument, revealing near contiguous density coming off the Kir6.2 subunit towards the cavity and with only a short gap to the density in the cavity, whereas the current manuscript shows what seems to be a more disembodied density in the cavity, and appears to be at no better resolution than that of Wu et al. Moreover, Wu et al. showed additional correlation in so far as there was no density in the SUR1-Kir6.2 fusion structure (in which the N-terminus is not free to act as an extended peptide).Please also comment on whether in the current structures there is any continuity between Kir6.2 and the density attributed to the N-terminus of Kir6.2 within the core of SUR1 , and what in the new structures makes it convincing that it is the N-terminus. The present functional data are not sufficient to make this conclusion.We regret to have given the reviewers the impression that we were being dismissive of the study by Wu et al. In response, we have revised the manuscript and provided a detailed account of the evidence described in their paper for the KNt cryoEM density (Results and Discussion sections) to properly acknowledge their findings.The paper by Wu et al. compared their GBC-bound cryoEM structures using the SUR1-Kir6.2 fusion construct with a 39aa linker, their previous GBC bound structure of channels formed by co-expression of SUR1 and Kir6.2 as separate proteins, and the GBC/ATP bound structures also of channels formed by individual SUR1 and Kir6.2 proteins we published. They noted cryoEM density in SUR1’s central cavity in structures of channels formed by separate SUR1 and Kir6.2 proteins but not by the SUR1-Kir6.2 fusion. More specifically, they used our published GBC/ATP structure to show near contiguous density coming off the Kir6.2 subunit towards the SUR1 cavity. They proposed the density likely corresponds to KNt based on previous structure-function data and argued the lack of density in the fusion cryoEM map is due to the large size of the linker that cannot be accommodated in the central cavity.We have now included additional cryoEM map figures showing that when our GBC/ATP and RPG/ATP-bound cryoEM maps are filtered to a lower resolution (6Å), contiguous density emanating from the first structured residue in Kir6.2 (R32), following along SUR1-L0, and reaching the ABC core central cavity is seen . While we agree Wu et al. offered a strong case of argument for the KNt density assignment and our own refined maps showing contiguous density further supports the assignment, there could be alternative structural interpretation given the low resolution of this density. For example, the cryoEM density of TMDs-NBDs linkers as well as the NBD1-TMD2 linker is largely missing in all the published structures and there is a possibility that the proposed Kir6.2 N-terminus density actually corresponds to these missing parts. For this reason, we decided to take the time to design biochemical and functional crosslinking experiments before rushing to publication.Although the reviewers felt that our functional data was not sufficient , we believe this data further bolsters the structural data and offer insight into the role of the KNt-SUR1 central cavity interaction in modulating channel activity . Thus, while the cryoEM structure, crosslinking-mass spectrometry, and functional crosslinking results individually do not provide definitive proof for the KNt density assignment, taken together they offer, in our view, compelling experimental evidence for the structural model proposed by Wu et al. and us.2) Address the weaknesses in the new experimental evidence for the binding of the KNt and its role in the pharmacochaperone effect:A) Potentially the strongest new evidence is the MS identification of cross-linked peptides including the KNt and the ABC core. However, there are major concerns that the XL-MS workflow used could produce false positives, particularly because of the absence of an alkylation step to reduce the chance of disulfide scrambling. These experiments should be repeated using an alkylation step and with tighter mass tolerances, using a well-established or validated XL-MS workflow.  Complete details of the experimental methods and the XL-MS raw data files should be included.It should also be acknowledged that the cross-linking is done on purified protein and not on channels in membranes, so it is possible the protein adopts conformations it might not normally do under physiological conditions.We thank reviewer 4 for his comments and offer the following explanations for our experiments. Although the method the reviewer recommended from the Heck lab is a good one, it requires a completely different informatics workup (XlinkX) and is better suited for the analysis of cross-links in more complex digests. The second paper from Lan Huang is a review of cross-linking, but also highlights the sulfoxide containing cleavable crosslinkers developed in their laboratory that was also used by the Heck lab. While a sulfoxide crosslinker could be used to repeat these experiments, and XlinkX software used to interpret results, we don’t feel that it’s necessary for analyzing crosslinks in a purified protein preparation such as ours. We respectfully disagree with the reviewer’s assessment that because we didn’t follow a “mainstream” method that our results are invalid. We and others have used ETD to break disulfide bonds in native proteins in many previous studies. As the disulfide bond in DSP is essentially identical to the disulfide bonds in peptides (-CH2-S-S-CH2-), there is no reason to believe that ETD wouldn’t also preferentially fragment the disulfide bond in DSP, and that is precisely what we have observed. The fact that we could perform the peptide identifications using a common search engine (Sequest) using MS3 data is a strength, not a liability. In fact, using MS3 based peptide identification in conjunction with a cleavable crosslinker is a common workflow when using a cleavable crosslinker, and this is outlined in Figure 2A of the Huang review.We also appreciate the reviewer’s concern about disulfide bond scrambling that may occur due to our omission of an alkylation step. This is certainly a concern when analyzing disulfides between cysteine residues in proteins. However, it does not pose a problem when using our crosslinking method with DSP, because it would not lead to misidentification of crosslinks. If a free cysteine carried out a disulfide exchange with a DSP crosslink, the resulting mass of the ETD liberated peptide would not lead to an identification, because it wouldn’t have a DSP remnant of 88 mass units. The worst case scenario would just be that we would have a loss of sensitivity if one of the DSP crosslinks underwent a disulfide exchange reaction with a free cysteine. We chose not to include the iodoacetamide alkylation to avoid over-alkylating the proteins, because the usual second addition of DTT that we normally do to scavenge the excess IAA after alkylation is complete couldn’t be done.Another one of the reviewer’s concerns was that we didn’t describe the cross-linking reaction in detail. We have now provided more experimental details on the reactions in Materials and methods.The reviewer is correct that the analysis of crosslinks using mass spectrometry is a demanding experiment. However, we feel that sufficient precautions were made to prevent false identification of the reported crosslinks. This is because: 1) the SEQUEST search of the two liberated peptides coming from a single MS2 event had to simultaneously identify unique peptides coming from either SUR1 or Kir6.2 when using a rat Swiss-Prot protein database containing over 8,000 entries, 2) both identified peptides had to have the +88 mass unit increase localized to a lysine residue due to the expected fragmentation of the disulfide bond within the DSP crosslinker, 3) the lysine identified at the crosslinking site had to exhibit a missed trypsin cleavage, 4) the identified peptides in the SEQUEST search had to have less than a 10 ppm mass error, and 5) the two identified peptides in the crosslink had to have a combined mass that matched the mass of the still crosslinked peptide detected in the MS survey scan. We apologize for the error in the mass tolerance of the precursor ions during the SEQUEST search previously given in the Materials and methods section. This has now been corrected.A suggestion was made to share the raw files so other groups have the option of validation. The raw files used for the crosslinking experiment has been deposited to the ProteomeXchange Consortium via the PRIDE repository .Finally, we have acknowledged in the Results section that the crosslinking is done on purified proteins and not on channels in membranes, so it is possible the protein adopts conformations it might not normally do under physiological conditions.On this question of false positive interactions, note that some of the SUR1-SUR1 crosslinking residues seems very far apart in both propeller and quatrefoil structures. For instance, K915 to K1412 looks to be ~70Å yet the linker is only 15A.We thank the reviewer for pointing this out. We have now added in the Materials and methods and figure legend that because we use detergent solubilized, purified channel proteins for crosslinking, we cannot rule out the possibility of inter-SUR1 crosslinks. When we interpret the cross-linking/mass spectrometry results we do keep this in mind. The SUR1 K915-K1412 crosslink is very likely a result of inter-SUR1 occurrence within the same channel. As noted by the reviewers, K915 and K1412 are very far apart in the single SUR1 protein in our structure. However, the residues from adjacent SUR1 subunits in the same channel could reach crosslinking distance if SUR1 subunits are flexible. In this regard, it is worth noting that we indeed observe flexibility of SUR1 subunits that deviate from C4 symmetry (Results section). While we cannot rule out inter-channel SUR1 crosslinks, the probability of detecting such random crosslinks would be expected to be quite low. An inter-channel crosslink is even less likely the case for the Kir6.2-K5 and SUR1-K602 pair considering that the Kir6.2 tetramer is surrounded by SUR1 subunits. Nevertheless, we cannot completely rule out the possibility that this crosslink arises from dissociated subunits.For the caveats discussed above we only used the CL/MS data as supporting evidence in our manuscript (see response to point 1 above). We note however, in our published study examining the effects of GBC and CBZ on photo-crosslinking of azidophenylalanine engineered at amino acid 12 or 18 position of Kir6.2 to SUR1, crosslinking was performed in intact cells. While we were unable to identify SUR1 residues to which Kir6.2-12AzF or 18AzF form crosslinks, our observation that crosslinking is enhanced by GBC and CBZ again provide supporting evidence of pharmacochaperone-dependent KNt interactions with SUR1.B) The electrophysiological tests of cross-linking of the Kir6.2-L2C mutant aim to provide functional evidence for KNt contacting the ABC core domain in an intact membrane under physiological conditions, however, in the opinion of the reviewers and the Reviewing Editor, these current results do not provide convincing support for the hypothesis. The text states that "co-expression of Kir6.2-L2C with WT SUR1 resulted in channels that displayed an accelerated current decrease in I2 (250µM) that was partially reversible by subsequent exposure to DTT […]" In fact, both wild type channels and Kir6.2-L2C/SUR1-C1142A channels show a similar extent of inhibition during iodine application. If the difference between Kir6.2_L2C and controls is in the rate of current decrease, there is no quantification to support this claim. It is true that there was no DTT reversal apparent in the controls, but this reversal is only apparent in a subset  of the Kir6.2-L2C records. Were the experiments that showed more profound reversal only the ones with small currents ? Was the effect of DTT alone tested on wild-type or mutant channels? As for the drug block experiments, how are the effects of iodine differentiated from the effects of rundown ?It is unclear how best the authors can address these concerns and shortcomings in the physiological cross-linking experiments. Possibly more experiments along these same lines will make the effects more clear. Alternatively, given the apparent distance between the putatively cross-linked sites in the structure, it may be helpful to investigate the effects of a rapid, non-zero-length crosslinker such as a bis-MTS reagent. Although it would be preferable to include an improved version of these experiments, if this is not possible then these experiments should probably just be omitted from the revised manuscript.2O2 or copper phenanthroline or even form disulfide bond spontaneously upon patch excision .We appreciate the reviewers’ comments. These experiments turned out to be more challenging compared to our previous crosslinking pairs in the cytoplasmic domain of the channel (including Kir6.2-E229C/Kir6.2-R314C and Kir6.2-Q52C/SUR1-E203C). Cytoplasmic cysteine pairs are easily crosslinked by H2O2 and copper phenanthroline at different concentrations as we did in prior studies and tested more than 20 SUR1-Kir6.2 cysteine pair combinations, however, no clear reversal of currents by DTT after exposure to oxidizing agents was ever observed. After reviewing membrane protein crosslinking literature, we learned that I2 works better for residues in the transmembrane regions that are less solvent exposed. Indeed, systematic testing of multiple SUR1/Kir6.2 pairs using a paradigm of I2 exposure followed by DTT exposure, we were finally able to see a clear DTT reversal effect on the currents of the SUR1-C1142/Kir6.2-L2C pair. As noted by the reviewers, not every patch showed clear reversal and those that did not tended to show less current reduction with I2 exposure. While we cannot offer definitive explanations, a likely reason is that I2 did not induce significant crosslinking in these patches. This could happen if the patches were deeply recessed and slow to respond to reagents present in the bath solution (this is not uncommon in inside-out recordings based on our >20 years of experience). The other possibility is that channels in those patches had higher open probability such that the probability of KNt reaching the SUR1 central cavity was low. Consistent with the latter explanation, when channels were exposed to KINT solution containing 1mM EDTA which significantly reduces rundown caused by Mg2+-dependent loss of PIP2 , we did not see rapid current decay in I2 nor subsequent reversal by DTT. This is the reason why we performed the crosslinking experiments in KINT without EDTA. Under this condition, WT channels display a variable degree of “rundown” as well appreciated by researchers in the field. To our knowledge what accounts for this variability is not well understood, which makes it difficult to differentiate rundown from I2-induced decrease of currents. In light of the reviewers’ comments we have removed the phrase “accelerated current decrease” and the revised statement now reads: "co-expression of Kir6.2-L2C with WT SUR1 resulted in channels that displayed current decrease in I2 (250μM) that was partially reversible by subsequent exposure to DTT […]".For the Kir6.2-L2C and SUR1-C1142 crosslinking, we had to test many different conditions. Initially we tried HWe agree with the reviewers that performing more experiments will not resolve the intrinsic difficulties of these crosslinking experiments. However, we respectfully disagree that the data presented should be removed as we believe that the DTT-induced current reversal (despite not in every patch) provides strong functional evidence for the structural interpretation and for a role of this interaction in channel activity. Taken as a whole, our structural, biochemical, and functional results converge and corroborate to support the mechanism we propose on how pharmacochaperones inhibit channel activity and correct channel trafficking defects. Nonetheless, we appreciate the weaknesses pointed out by the reviewers and have tried our best to revise the manuscript to explain the crosslinking experiments more clearly .Finally, regarding testing the effect of DTT alone on WT or mutants, we have documented such control experiments in our previous publications . As such, we do not feel the need to show these recordings again. We have now included in the Figure 5 legend to specifically state that DTT alone does not cause significant channel run-up or run-down aside from an acute reversible blocking effect as indicated by thearrow in the first recording  and cited our published papers where this was documented.C) Although deletions at the N-terminus of Kir6.2 are said to abolish the pharmacochaperone effect (by interfering with the putative interaction of the KNt with the drug bound within the SUR core), there is a very clear pharmacochaperone effect on the ΔN5 mutation in Figure 4—figure supplement 1C (and data are not shown for the ΔN10 mutation). If indeed the KNt deletion prevents the interaction with the SUR-bound drug, it suggests that the pharmacochaperone effect may be independent of the KNt interaction. The weaker (but still not absent) pharmacochaperone effect with the addition of the F27S mutation (panel B) may simply indicate that the KNt deletion destabilizes the protein and the F27S destabilizes it further, with both effects independent of the drug binding. At a minimum, the results with ΔN10 (with no F27S) should be shown, and this alternative interpretation should be discussed.As requested we have now included a blot of WT-SUR1 co-expressed with ΔN10-Kir6.2 and treated with vehicle, glibenclamide, repaglinide, or carbamazepine  showing a further attenuation of drug effects on the maturation of WT-SUR1, similar to that seen for F27S-SUR1.Regarding the alternative interpretation that KNt deletions make SUR1 less stable and F27S makes the protein even less stable, our response is as follows. By looking at the SUR1 upper band intensity, this may seem like a logical alternative interpretation. However, an important reminder here is that the upper band only appears when the channel complex is fully formed and has passed the ER quality control, which requires successful assembly of SUR1 with Kir6.2. Thus the reduced upper band indicates less stable SUR1/Kir6.2 complex and not SUR1 alone. We think it is unlikely that KNt deletion significantly impacts SUR1 stability as this would be expected to lead to increased ER associated degradation and decreased lower band intensity, which is not what we observed How do we explain the residual drug effects seen in SUR1 co-expressed with ΔN5- or even ΔN10-Kir6.2? We believe the most logical interpretation is that the distal ~20 amino acids of KNt is critical for SUR1 and Kir6.2 interactions during the initial assembly process even in the absence of drugs. Drug binding boosts the interaction in part by participation of KNt in drug binding but also by stabilizing SUR1 in an inward-facing conformation that allows KNt to interact with the central cavity for channel assembly. In addition, we have previously shown using metabolic pulse-chase experiments that drug binding slows ER degradation of mutant SUR1 , which would allow more time for Kir6.2 to interact before the mutant SUR1 is targeted for ERAD. Progressive deletion of KNt leads to a gradual loss of PCs’ ability to chaperone channel assembly by making KNt an increasingly weaker anchor. We have now revised the Results and Discussion sections and the cartoon figure  to explain our data more clearly.Po, it can give the impression that their expression is not affected.Reviewer 2 also commented that in our study deletion of Kir6.2 N-terminus markedly reduced maturation of WT SUR1; however, previous studies co-expressing WT SUR1 and Kir6.2Δ5 or Kir6.2Δ10 showed descent currents. We note that using oocyte or mammalian cell transient expression, it is always possible to obtain membrane patches containing sufficient channels from individual cells that do not reflect the overall expression level. Also, because channels lacking the Kir6.2 N-terminus have higher 3) In Figure 3—figure supplement 1B,C, it is not possible to deduce if the mutations in SUR1 had any effect on drug block because it is very hard to distinguish current rundown from block in the records provided. The difficulty is even greater when the currents are very small as for the mutant channel. How was the% current measured in panel D? There really seems to be no steady state block by the drug  and there is no clear, rapid onset of block. The problem with these drugs is that they are largely irreversible and they block slowly at low concentrations. It would be better to test a single drug concentration (preferably 100nM) on each patch. Alternatively, the S1238Y mutation of SUR1 could be exploited for comparisons as sulfonylureas block these mutant channels reversibly and at lower affinity.The W1297 functional testing experiments were meant to provide further support to our drug binding site structural model. However, in revising the manuscript, we believe this is not necessary and may cause more confusion (based on reviewers’ feedback). As such, we have removed this supplementary figure.For reviewers’ information, we have used a similar experimental scheme (10 and 100nM GBC) to study other binding pocket mutations in our recent publication where a number of mutations but not all render GBC inhibition highly reversible .4) The value of the Rb+ efflux experiments in Figure 3 is unclear and the conclusions drawn from these experiments may be overstated. The drugs were applied for up to 40 min. Is this long enough to affect surface trafficking of SUR1? Can it can be concluded if the data primarily represent the effect of the drug on channel inhibition? The authors argue that differences between mutational effects on the ability of repaglinide and carbamazepine to inhibit flux confirm that the mutations are directly in the drug binding site and do not have an indirect-allosteric effect. At first glance, the effects of mutating these residues in the binding pocket on the ability of the two drugs appear to be similar . Is flux a sensitive enough measurement to make such distinctions? Why were mutations tested using Rb+ efflux, rather than patch clamp? Efflux is both determined by the number of channels in the membrane and by the ability of the drug to block the channel. So they can't state 'mutations reduced the sensitivity of the channel to inhibition' – unless they have shown that changes in surface expression do not occur on the time scale of their experiments.Although the experiments were conducted over a 40 min period, the difference in efflux rate was already apparent during the first few minutes when we look at the efflux profile over time . We don’t have any experimental evidence that the number of channels does not change over the course of the efflux experiment in the present study for every mutant. However, our previous surface biotinylation studies have not found significant changes in surface expression of WT channels in COS cells over 30min .In our previous publication where we tested the effect of GBC binding site mutations on the ability of GBC to inhibit channel activity using Rb efflux assays, we did include an Rb efflux profile example in addition to summary data. In the current study we repeated these experiments using the same set of mutations but testing response to RPG and CBZ with the intent to further validate our structural models for the RPG and CBZ binding pockets. We therefore only presented summary data and referred to our previous publication for the initial GBC experimental dataset. Rb efflux is an efficient way to assess drug response. In our previous study we used both Rb efflux and electrophysiology data to demonstrate effects of binding site mutations . As we believed the main point of this experiment is to show reduced inhibition by the mutants to confirm the binding pocket model, we did not think it is necessary to perform electrophysiology experiments especially considering the focus of this paper is on the pharmacochaperoning effects of the drugs. In place of electrophysiology, we performed pharmacochaperone rescue experiments to show that binding pocket mutations also compromised PC rescue effects.In light of the reviewers’ comments, we have removed the Rb efflux results in Figure 3 and only show the impact of the binding site mutations on chaperoning effects of the drugs. Note, we also replaced the original Western blot showing the effect of RPG with a new one showing the effect of GBC and RPG side-by-side to facilitate direct comparison .[Editors’ note: the author responses to the re-review follow.]We appreciate the valuable new data in the paper, but our expert mass spectrometry reviewer still has serious concerns that the MS crosslinking results are too prone to false positives, without alkylation to prevent disulfide scrambling and a higher m/z resolution analysis of the crosslinking products. Given this concern combined with the weaknesses already pointed out in the functional disulfide crosslinking work, it seems that the central contention of the paper about the interaction between the KNt and the SUR1 core is not adequately supported by the experimental results.We understand that you may disagree with these concerns, but we consider them too important to overlook. At this point, we are rejecting the submitted manuscript, but we would welcome a new submission of a manuscript that addressed these concerns, most likely with new experimental results. Alternatively, if you think that we have just got it wrong, our rejection of the paper frees you to submit the paper to other journals.Reviewers #1 and #3 were satisfied with the revisions.Reviewer #4:[…] The authors also use DSP in a way that it has never been used (I asked them to include a reference to their workflow and they didn't). DSP is usually chemically reduced before MS experiments (and compared to a non-reduced experiment). However, these authors extended Huang's concept of the "MS-cleavable" crosslinker from Collisional activation (CID) acting upon a sulfoxide bond , to ETD acting upon disulfides . Prior to using such a workflow to reach biologically significant decisions, there are a number of hoops authors jump through, one being a methods development study where they show a histogram of the length of the ID'd crosslinks (where the 70 Å cross-link they ID'd would have been a problem). Here I invited them to prove their workflow on their engineered disulfide bond peptides, which like all other of my requests, they declined.And the authors don't really detect half-DSP conjugated lys residues- they detect peptides that happen to have modifications that sum to 88 +/- 1 Da (and there are a lot of ways to get to that mass).They use the insoluble crosslinker, rather than water soluble analogue.Unlike the studies of established labs, they don't bother using isotope labeled DSP to validate their results. These are commercially available.Most of these concerns are in my previous review.We thank Dr. Agar for his constructive criticisms and valuable suggestions to improve our XL-MS experiment. Two fatal design flaws were pointed out, one concerns potential false positives due to disulfide scrambling, and the other is the 1Da MS/MS cut-off. On the second point, we apologize for not making this clear in our R1 version of the manuscript. The 1.0 Da value is for the MS3 scan in the Orbitrap Fusion’s ion trap and is within the setting commonly used , while the precursor tolerance is set at 10 ppm.Nonetheless, given the reviewer’s serious concerns about our results and that many validation steps were requested to support our data which will take significant amount of time, we decided to repeat the XL-MS experiment using a different crosslinker that has been well documented in a number of published studies.Specifically, we used an isotopically coded, CID-cleavable, biotinylated amine-reactive homobifunctional crosslinker, CBDPS (14Å space arm) (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033670/) for our experiments. The purified proteins were reduced with DTT and alkylated immediately after crosslinking and before trypsin digestion. The digested peptides containing the crosslinker were affinity purified via the biotin tag and MS analysis was conducted with mass precision tolerances of 5 and 10 ppm for precursors and fragment ions, respectively .Below are some examples of published studies which have used CBDPS with similar workflow to derive protein structural information:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814369/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3642325/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501500/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561701/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453469/The new experiment identified 2 inter-protein peptides between Kir6.2 and SUR1 and 4 intra-protein peptides in SUR1 as listed below , with the distance between α carbons of the two cross-linked lysine residues in our model indicated where possible.Kir6.2-SUR1:Kir6.2 K5-SUR1 K205: 40.7Å? (see discussion below)Kir6.2 K5-SUR1 K602 (identified in the previous DSP XL-MS experiments): 17.6ÅSUR1-SUR1:K276-K394: 21ÅK276-K644: K644 is not modeled in the structure but is predicted to be located below K276K941-K948: in NBD1-TMD2 linker not modeled in the structure but should be close given residue numbersK1344-K1412: 17.6ÅThe crosslinks identified using CBDPS are not identical to those identified in our previous experiments using DSP . Importantly, however, the same crosslinked peptide linking Kir6.2-K5 and SUR1-K602 was identified in both experiments . The new XLMS results validate the physical proximity between Kir6.2-K5 and SUR1-K602 in our purified channels under the stated experimental conditions and strongly support our assignment of the Kir6.2 distal N-terminus density in SUR1’s ABC core central cavity. Note, in addition to the crosslink between Kir6.2-K5 and SUR1-K602, we also identified another crosslink between Kir6.2-K5 and SUR1-K205. According to our dominant class of GBC-bound cryoEM structure the Kir6.2 N-terminus is modelled within the SUR1 ABC core central cavity such that the distance between Kir6.2-K5 and SUR1-K205, which is in the proximal L0 region close to the Kir6.2 tetramer would be quite far (40.7Å). However, we should point out that in our cryoEM map, Kir6.2 N-terminus density is relatively poorly resolved compared to the rest of the structure, an indication of its flexibility even in the GBC-bound state. Thus, the identification of this alternative crosslink is consistent with our overall cryoEM structure data and suggests Kir6.2 N-terminus could dissociate from the SUR1 central cavity such that Kir6.2-K5 is within reach of SUR1-K205 via the CBDPS crosslinker.Because the potential problems raised by the reviewer on our previous DSP crosslink experiments, we have deleted the original figure and methods and replaced them with the new CBDPS XLMS result figure and methods. The raw data have been deposited into the proteomic database PRIDE.