A KATP channel fusion construct for cryo-EM structure determination
To overcome the compositional heterogeneity of the KATP channel when heterologously co-expressing SUR1 with Kir6.2 (Li et al., 2017), we created a SUR1-Kir6.2 fusion construct, in which the intracellular C-terminus of SUR1 is covalently linked to the N-terminus of Kir6.2 by a linker (Fig. S1A). Fusion construct with a 6 amino acids (6AA) linker was previously used to elucidate the association and stoichiometry of KATP channels (Clement et al., 1997) and was recently used to solve the structure of KATP channel in complex with Mg-ATP and Mg-ADP (Lee et al., 2017). To avoid potential artifacts on the structure of the KATP channel because of limited linker length, we used a flexible linker with 39 amino acids (39AA), which should be sufficient for a 130 Å linear distance when fully extended. Similar to the wild-type channel, the fusion construct can be inhibited by ATP and activated by Mg-ADP in an inside-out patch clamp, which is the characteristic property of KATP channel (Fig. S1B). However, inhibition by GBM is markedly decreased (Fig. S1B). Because it has been reported that the individually expressed SUR1 subunit is sufficient for high affinity GBM binding (Aguilar-Bryan et al., 1995) and our construct has an intact SUR1 subunit, we reasoned that the reduced GBM inhibition is due to insufficient coupling between SUR1 and Kir6.2 of our fusion construct, as discussed later. To be noted, it is reported that 1 µmol/L GBM can fully inhibit the 6AA linker construct in whole cell 86Rb+ efflux assays (Shyng and Nichols, 1997), this is probably because the GBM can bind SUR1 to counteract the activation effect of intracellular Mg-ADP or Mg-ATP, rather than directly inhibits the 6AA fusion channel. It is also reported that 300 µmol/L tolbutamide can attenuate the currents of 6AA construct in inside-out patch clamp recording (Lee et al., 2017). This is possibly due to the low affinity block of sulfonylurea on Kir6.2 channel (Reimann 1999). Whether the 6AA construct can be directly inhibited by the GBM bound on SUR1 remains to be thoroughly determined.
Structures of the KATP channel in the ATP + GBM and ATP states
We aimed to obtain structures of KATP with and without GBM at higher resolution than our previous GBM state (Li et al., 2017), which can be used to locate the GBM density by direct comparison. Our previous study demonstrated that the KATP channel displays a large degree of conformational heterogeneity when in complex with the high affinity inhibitor GBM alone (Li et al., 2017). The additional ATP molecule, an endogenous inhibitor of KATP, can further stabilize the Kir6.2 subunit to reduce conformational heterogeneity (Martin et al., 2017b). Therefore, we applied ATPγS, a slowly hydrolyzable and functional ATP analog, to stabilize the structures of the KATP channel with and without GBM (Schwanstecher et al., 1994a).
The fusion channel protein was purified and then subjected to cryo-EM single particle analysis (Fig. S1C and S1D). The structure of KATP in complex with ATPγS and GBM was solved at an average resolution of 4.3 Å, slightly higher than the structure in complex with ATPγS alone at a resolution of 4.5 Å (Figs. 1A–C, S2, S3A, and Tables S1 and S2). The overall KATP channel structure and SUR1 conformation in the ATP + GBM state is almost identical to that in the ATP state based on our density maps (Fig. S3B). Therefore, we focus the discussion on the structure of the ATP + GBM state with a higher resolution, unless stated otherwise.
Focused three-dimensional (3D) classification on the Kir6.2 cytosolic globular domain (CTD) isolated two major classes with distinct conformations (Fig. S2 and Table S1). The CTDs of one class have relative rotation to the other, as observed previously in the structure of the ATP + GBM state (Martin et al., 2017b). Each class was individually refined to reach resolutions of 4.3 Å or 4.6 Å. In contrast, the transmembrane domain (TMD) of the two classes shares the same structure and focused refinement with all particles reached an overall resolution of 4.1 Å (Fig. S2C and Table S1). The maps demonstrate side chain densities for most residues and enable further improvement of our previous model based on medium resolution maps. The overall structures of both classes are similar to the previous symmetric Class 1.1 structure of the GBM state (EMD-6689) (Li et al., 2017) or that of the ATP + GBM state (EMD-8470) (Martin et al., 2017b), with the central Kir6.2 in a closed state and the peripheral SUR1 in an inward-facing inactive state (Fig. 1B and 1C). By comparing the structures of the two classes, we found that the CTDs of Kir6.2 (R32 to L66 and K170 to L356) move in a rigid body fashion relative to the transmembrane domains, resulting in a 3.9-Å downward movement and 13.2° clockwise rotation (Fig. 1D and 1E). In contrast, the structure of transmembrane domain of Kir6.2 and SUR1 is largely unchanged (Fig. 1D and 1E). Therefore, the interface between Kir6.2 CTD and SUR1 TMD0-L0 fragment is altered and the conformations of the linkers between the two rigid bodies also change. Here, we refer to class 1 structure as the “T state” (the tense state) and class 2 as the “R state” (the relaxed state). Specifically, in the “R state” structure, the linker between βA and the interfacial helix (IFH) of Kir6.2 moves away from the TMD0-L0 of SUR1 (Fig. 1F). The differences between the two conformations described herein are not due to differences in ATPγS occupancy on CTDs, because we observed similarly strong ATPγS densities in the maps of both classes (Fig. S4A). Instead, they possibly reflect the endogenous mobility of the CTDs in the Kir6.2-inhibited state, because we observed similar conformational heterogeneity of the KATP channel in the ATP state and the Mg-ADP state as described later. The co-existence of “T state” and “R state” might be functionally important since the rotation of CTD relates to channel gating in another Kir channel member GIRK2 (Whorton and MacKinnon, 2011, 2013) .
GBM binding site on SUR1
Previous studies demonstrated that mutations of Y230A and W232A on the lasso motif reduce GBM affinity (Vila-Carriles et al., 2007), indicating the lasso motif might be in proximity to or on the binding site of GBM. Therefore, we compared the density maps around lasso motif between the ATP state and the ATP + GBM state, and found that the extra density near the lasso motif in the previous medium resolution map is present in both states. With improved resolution and map quality, we found that this extra density was contributed by both the M5-Lh1 loop and an unknown ligand, probably a lipid head group that constitutively binds KATP, because it is also present in the Mg-ADP state described later (Fig. S4B). Instead, a previously unmodeled density inside the SUR1 central cavity in the ATP + GBM state map has a distinct elongated shape that matches the GBM molecule in an extended conformation (Figs. 2A and S4C–E). In contrast, this density is not present in the ATP state, where GBM was not supplied into the sample (Fig. 2B). This unambiguously demonstrates that the extra density inside SUR1 central cavity in the map of the ATP + GBM state represents the GBM molecule (Figs. 2A and S4C–E). The extended conformation of GBM bound to SUR1 is dramatically different from the compact conformation of GBM bound to human aldo-keto reductase 1C3 subfamily (AKR1Cs) (Zhao et al., 2015) (Fig. S4C). Our results confirmed the GBM binding site reported by another group recently (PDB: 6BAA, EMD-7073) (Martin et al., 2017a). Accompanied by mutagenesis results, they suggested indirect allosteric effects of Y230A or W232A. These mutations on the lasso motif might change the structure of adjacent transmembrane helices that are essential for GBM binding (Martin et al., 2017a). Therefore, mutagenesis results might be affected by allostery, and comparison between the maps of KATP solved from samples supplied with and without GBM is essential to definitively assign the non-protein density for GBM.
Location of Kir6.2 N-terminus
The fact that we observed strong GBM density inside SUR1 further reinforced our above assumption that SUR1-Kir6.2 fusion protein can bind GBM but the reduced GBM inhibition is because of insufficient coupling between SUR1 and Kir6.2 due to the covalent linker (Fig. S1B). When docking the improved model of SUR1 and GBM into our previous Class 1.1 map of the GBM state (EMD-6689) (Li et al., 2017), we found an additional positive density in the SUR1 central cavity that originates from the proximity of the GBM molecule and extends out of the SUR1 ABC transporter module in the direction of Kir6.2 CTD (Fig. 2C). Our previous GBM state structure (Li et al., 2017) and the ATP + GBM state structure by another group (Martin et al., 2017b) were solved using KATP protein generated by the coexpression of SUR1 with Kir6.2 as separated polypeptides; therefore, the Kir6.2 protein had a native N-terminus in these studies. In the map of the ATP + GBM state (EMD-7073), we observed a similar density (Fig. 2D and 2E). Unfortunately, because of limited local map quality, we cannot infer the identity of this density purely from these maps. In contrast, we did not observe any density at the same position in our map of the ATP + GBM state that was solved using the SUR1-Kir6.2 fusion construct, in which the C-terminus of SUR1 is covalently connected to the N-terminus of Kir6.2 by a linker (Fig. 2F) and the linker together with Kir6.2 N-terminus is completely disordered in our cryo-EM map. In our previous models (PDB: 5WUA) (Li et al., 2017), which were built according to the cryo-EM map, only one amino acids of the SUR1 C-terminus were unmodeled, and they are unlikely to contribute to such an elongated unaccounted density. Therefore, this positive density, which is close to the GBM binding site, might represent the N-terminus peptide of Kir6.2 with 30 amino acids omitted in the model. This observation agrees with previous biochemical data showing that Kir6.2 can be labeled by 125I-azidoglibenclamide and deletion of KNtp reduced not only 125I-azidoglibenclamide labeling (Babenko and Bryan, 2002; Vila-Carriles et al., 2007) but also the affinity for GBM and repaglinide (Kuhner et al., 2012). In addition, GBM enhanced the cross-linking of SUR1 with Kir6.2 with the photo cross-linkable amino acid incorporated in the N-terminus (Devaraneni et al., 2015), indicating that the KNtp should be in proximity to SUR1. This is further supported by the electrophysiological data that high affinity inhibition of tolbutamide, GBM and repaglinide to the KATP channel were either abolished or markedly decreased when KNtp is deleted (Devaraneni et al., 2015; Kuhner et al., 2012; Reimann, 1999). Moreover, the density of the putative KNtp is closed to the 1-chloro-4-methoxybenzene group of GBM (Figs. 2A and S4E), the so called “B” site, where the azido group in125I-azidoglibenclamide sits. This is in accordance with previous findings that the 125I-azidoglibenclamide but not 125I-glibenclamide can label Kir6.2 subunit (Aguilar-Bryan et al., 1990; Schwanstecher et al., 1994b). Because the length of KNtp is conserved and limited (Fig. 2G), the special position of the Kir6.2 N-terminus intuitively suggests that it attenuates channel gating by acting as a chain to restrain the rotation of CTD, which is important for Kir family channel gating (Whorton and MacKinnon, 2011, 2013). To further validate this model, we inserted one to three Gly-Ser dipeptides (GS) between amino acid R27 and T28 of Kir6.2, a position before the structured Kir6.2 CTD and not conserved in the primary sequence (Fig. 2G), suggesting this position is not directly involved in SUR1 binding. We hypothesized that the KNtp of these mutants are still able to bind SUR1 but the restriction of the CTD rotation by KNtp might be reduced. Indeed, we found that the inhibitory effect of GBM progressively becomes weaker as the linker length increases (Fig. 2H). The SUR1-Kir6.2 fusion construct demonstrated little GBM inhibition (Figs. 2H and S1B), a phenotype similar to the KATP channel assembled with KNtp deleted Kir6.2, suggesting the KNtp in the fusion construct cannot bind SUR1 anymore, because of the covalent linker. Moreover, without sulphonylureas, truncation of KNtp increases channel activity and reduces ATP sensitivity (Babenko et al., 1999; Koster, 1999; Reimann, 1999) and synthetic KNtp dose-dependently attenuates ATP inhibition (Babenko and Bryan, 2002), suggesting KNtp might remain bound to SUR1 when ATP, but no sulphonylurea, is present, albeit with decreased affinity. When we superposed our SUR1 ABC transporter module model of the GBM + ATP state to that from the another group (PDB: 6BAA) (Martin et al., 2017a), we found that there is a small but noticeable change of overall conformation of SUR1 and the angle between M10 and M16 is 42.7° in our structure whereas 39.3° in their structure (Fig. S4F). Whether the observed difference of SUR1 conformation is due to KNtp binding discussed above or different data processing procedure still waits further investigation.
Mg-independent ATP binding site on SUR1 NBD1
During cryo-EM data processing, 3D classification demonstrated continuous conformational heterogeneity of SUR1 in the ATP + GBM state. Specifically, four peripheral SUR1 subunits wobble around the central Kir6.2 channel in small angles. This greatly limited the resolution of the overall cryo-EM density map and deteriorated local map quality of the peripheral SUR1 ABC transporter module. Further improvement of SUR1 map quality at the ATP + GBM state benefited from focused refinement with local search on symmetry-expanded (Zhou et al., 2015) and signal-subtracted particles of the SUR1 ABC-transporter module (Bai et al., 2015). The map of “the isolated” SUR1 ABC-transporter module reached a resolution of 4.4 Å and demonstrated markedly enhanced quality, especially for NBDs (Fig. S2C). The map also suggests a register shift of residues 1,061–1,079 on M13 in the structure by another group (PDB: 6BAA) (Martin et al., 2017a). More interestingly, we observed extra density on the degenerate site of SUR1 NBD1. The shape and size of this density matched that of ATPγS (Fig. 2I). Moreover, this density was at the expected location of nucleotides in the nucleotide-bound NBD structure of other ABC transporters (Zhang et al., 2017). Therefore, we modeled this density as an ATPγS molecule. We observed a similar density in the map of the ATP state structure (Fig. 1A). In the sample preparation procedure, we added 2 mmol/L ethylenediaminetetraacetic acid (EDTA) to avoid the activation effect of ATPγS by chelating divalent ions (Proks et al., 2010). Therefore, we observed an Mg-independent ATP binding site in the degenerate site on the NBD1 of SUR1. It is possible that in the ATP + GBM structure reported by another group (Martin et al., 2017a), ATP molecule is also bound on the NBD1, but limited local map quality (EMD-7073) precludes any conclusion.
Architecture of the KATP channel in the Mg-ADP bound state
It is previously reported that KNtp is not necessary for channel opening and activation by Mg-ADP (Reimann, 1999). The fact that our 39AA fusion construct can be re-activated by Mg-ADP (Fig. S1B) suggests this construct can be used for structural study on the Mg-ADP activation mechanism. To trap SUR1 in an NBD-dimerized conformation, we supplemented the KATP fusion protein with Mg-ADP, the endogenous activator of the KATP channel. We also added NN414, a SUR1 specific KCO; VO43−, an ion that mimics the post-hydrolytic form of ATP γ-phosphate; and 08:0 PI(4,5)P2 lipid, a soluble PIP2 analogue which is required for KATP channel activation. The structure of KATP channel in the ADP bound state was solved to an overall resolution of 4.2 Å (Fig. S5 and Table S3). We observed similar “T state” and “R state” conformational heterogeneity of the Kir6.2 CTD and SUR1 ABC transporter module as in the ATP + GBM state or the ATP state. The map quality of specific regions was dramatically improved by focused classification and refinement (Fig. S5 and Table S3).
The overall architecture of KATP in the ADP bound state is also similar to the ATP + GBM state or the ATP state (Fig. 3A and 3B). Four Kir6.2 channel subunits are located at the center, and four SUR1 subunits are in the periphery. The SUR1 ABC transporter module connects to the Kir6.2 via SUR1 TMD0-L0 fragment. This is in great contrast to the “quatrefoil form” structure of Mg-ATP and Mg-ADP (Lee et al., 2017) which has a disordered lasso motif and a large rotation of the SUR1 transporter module. The “quatrefoil form” might represent a “decoupled” state, in which the conformational change of SUR1 cannot be transferred to Kir6.2 by either the TMD0-L0 or the N-terminus of Kir6.2. Our overall structure is similar to the coupled “propeller form” (Lee et al., 2017), which has an ordered lasso motif, despite some positional shift of SUR1 subunits (Fig. S6A and S6B). The large difference between the “quatrefoil form” and the “propeller form” might arise from different sample preparation procedures, because detergents were replaced by amphipols in their experiments (Lee et al., 2017). Similarly to the ATP or ATP + GBM state, the CTD of Kir6.2 in “T state” has a clockwise rotation (~11.8°) relative to that of “R state” (Fig. 3C). The two nucleotide binding sites on SUR1 are occupied by Mg-ADP, which results in an NBD-dimerized conformation (Fig. 3A and 3B). Moreover, the ATP binding sites of Kir6.2 are also occupied by ADP molecules, and the Kir6.2 channel is in the closed conformation, similarly to the ATP + GBM state (Figs. 3A, 3B and S6C), which is consistent with the result that ADP can inhibit the KATP channel albeit at a lower affinity (Hopkins et al., 1992; Schwanstecher et al., 1994a). The ADP binding mode at the inhibitory site of Kir6.2 is similar to that of ATP (Martin et al., 2017a) or ATPγS (Fig. S6C). Because ADP lacks the γ phosphate, there is no direct interaction between ADP and the γ-phosphate-coordinating residue R50 (Martin et al., 2017b). This explains why ADP binds Kir6.2 with a lower affinity and the R50 mutation shifts the IC50 curve to a similar range of ADPβS on wild-type channels (Schwanstecher et al., 1994a; Shimomura et al., 2006). We did not observe any density at the PIP2 binding site, probably due to the low affinity of the 08:0 PI(4,5)P2 to the purified protein and also the negative cooperativity of inhibitory nucleotide binding and PIP2 binding on Kir6.2 (Baukrowitz et al., 1998; Hilgemann and Ball, 1996; Shyng and Nichols, 1998). This is in accordance with the fact that we observed a closed state Kir6.2 structure in the Mg-ADP state, because PIP2 is necessary for channel opening (Baukrowitz et al., 1998; Shyng and Nichols, 1998).
Asymmetric NBD dimer of SUR1 in the Mg-ADP bound state
In our focused refined map of SUR1 in the Mg-ADP state, we observed the densities of Mg-ADP molecules in both the degenerate and consensus sites (Fig. 4A–C). We did not observe densities of VO43−, correlating with the fact that VO43− do not markedly affect SUR1 activity (Fig. S1B) (Proks, 1999; Shyng et al., 1997; Ueda et al., 1997). The Mg-ADP molecule in the degenerate site interacts with residues from both NBDs to induce a full closure of the degenerate site (Fig. 4C and 4D). In contrast, the Mg-ADP molecule in the consensus site primarily interacts with NBD2, and the consensus site is open (Fig. 4C and 4E). We use the distance between the Cα atoms of the conserved glycine on the ABC Walker A motif and the serine residue (a cysteine on SUR1 degenerate site) on the signature motif as an indicator of NBS closure. The distance between Cα of G1485 and C717 in the degenerate site is 11.5 Å, while the distance between Cα of G833 and S1383 in the consensus site is 13.0 Å (Fig. 4F). These distances are the opposites of the NBD-dimerized state structure of CFTR with Mg-ATP bound (PDB: 5W81) (Zhang et al., 2017). For CFTR, the degenerate site is partially open, and the consensus site is fully closed. The distance between G1350 and S461 in the degenerate site is 12.7 Å, while the distance between G550 and S1249 in the consensus site is 10.6 Å (Fig. 4G). The Mg-ADP state structure has the same asymmetric NBD-dimerized conformation as the Mg-ATP&ADP state structure reported by another group (PDB: 6C3P EMD-7339) (Lee et al., 2017). In particular, Mg-ADP molecule induces similar full closure of degenerate site compared to the Mg-ATP molecules (Fig. S6D).
Conformational changes of SUR1 ABC transporter module upon Mg-ADP binding
By aligning the TMD0 domain of SUR1 between the ADP and ATP + GBM states, we found a large conformational change of the SUR1 ABC transporter module (Fig. 5A). The full closure of the degenerated site induced by Mg-ADP binding leads to the asymmetric dimerization of two NBDs (Fig. 5B). These conformational changes are further conveyed to the transmembrane domain via coupling helices that interact with NBDs to drive two halves of the transmembrane domain of SUR1 ABC transporter module to move closer (Fig. 5C). Specifically, M14 and M17 move toward M8 and M11 (Fig. 5C). As a result, SUR1 central cavity shrinks, and the GBM and KNtp sites are disrupted. We added NN414, a KCO, to our sample (Fig. S7A). We observed two positive densities in SUR1. One is close to the top of SUR1, surrounded by M10-11 from TMD1, and M12 and M17 from TMD2 (Fig. S7B). The other is at the central cavity surrounded by M8 from TMD1, and M15–17 from TMD2 (Fig. S7C). Both of the densities are absence in the Mg-ATP&ADP state map, where NN414 was not added into the sample (EMD-7338, PDB: 6C3O) (Fig. S7B and S7C). By comparing the structures of transmembrane domain with (Mg-ADP state) and without NN414 (Mg-ATP&ADP state), the ligand corresponding to density 1 introduces local structural changes of M9-11 (Fig. S7D). But whether these densities represent NN414 needs further studies. Notably, we did not observe any density that might account for KNtp. Both our Mg-ADP state structure reported here and the Mg-ATP&ADP state structure reported by another group (Lee et al., 2017) are captured using the fusion construct, where the authentic KNtp is not present. It is possible that the KNtp might adopt an unknown conformation that is different from the one observed in the ATP + GBM state but compatible with our current Mg-ADP state structure.
Nucleotide binding induced NBD dimerization of SUR1
In the ATP and ATP + GBM state structures, SUR1 is in an inward-facing conformation. We observed ATP density only in the NBD1 degenerate site but not in the NBD2 consensus site, which suggests that the degenerate site has a higher affinity for Mg-free ATP, and ATP alone is not able to drive the dimerization of SUR1 NBDs. This correlates with previous studies that NBD1 of SUR1 can bind 8-azido-ATP with high affinity in an Mg-independent manner (Matsuo et al., 1999a; Matsuo et al., 1999b; Ueda et al., 1997). In addition, the subsequent binding of ATP to NBD1 can be strongly antagonized by prebound Mg-ADP (Ueda et al., 1997), indicating that the degenerate site of NBD1 can be occupied by Mg-ADP as well. Indeed, in our Mg-ADP state structure, we observed Mg-ADP molecules in both the NBD1 degenerate site and the NBD2 consensus site.
The asymmetric NBD dimer conformation observed in the Mg-ADP state structure suggests that the Mg-ADP molecule in the closed degenerate site directly mediates the formation of the NBD dimer interface, because this Mg-ADP molecule physically interacts with both NBDs. In contrast, the Mg-ADP molecule in the open consensus site interacts only with NBD2 but not with NBD1, indicating that it does not contribute directly to NBD dimer formation. By comparing the structures of individual NBD monomer in different states, we found a noticeable conformational change in NBD2 but not in NBD1 (Fig. 5D). Induced conformational changes of the NBD by nucleotide binding were previously observed in the crystal structures of isolated NBD of ABC transporter MJ1267 (Karpowich et al., 2001) and in the cryo-EM structure of CFTR (Zhang and Chen, 2016; Zhang et al., 2017) and also were sampled in MD-simulations (Jones and George, 2017). The conformational changes of NBD2 observed here might partially be due to the Mg-ADP binding and full closure of the degenerate site, but we suggest that the Mg-ADP binding on the NBD2 consensus site plays a more prominent role. This is supported by the previous radioactive 8-azido-ATP photo cross-linking results. First, the NBD2 consensus site mutation K1385M greatly reduces the ability of prebound Mg-ADP to prevent further ATP binding on NBD1, when Mg-ADP and ATP were added to SUR1 sequentially (Ueda et al., 1997; Ueda et al., 1999), suggesting that Mg-ADP acts on the NBD2 consensus site to allosterically increase the affinity of Mg-ADP on the NBD1 degenerate site. Second, Mg-ADP or Mg-ATP binding on the NBD2 consensus site can stabilize prebound ATP on the NBD1 degenerate site (Ueda et al., 1999), indicating that nucleotide binding on the NBD2 consensus site can send a signal to the degenerate site to increase its affinity for nucleotides, probably via the conformational change of NBD2, as we observed here.
Model for SUR1 conformational change
On the basis of experiments and structures of KATP channels in complex with different ligand combinations solved with different constructs, we propose the following hypothetic model for the conformational change of SUR1 subunit (Fig. 6A–H). When GBM is bound, GBM and the Kir6.2 N-terminus cooperatively bind inside SUR1 to inhibit channel gating (Fig. 6A and 6E). Without GBM, the Kir6.2 N-terminus can still bind to the central cavity of SUR1 to inhibit channel gating but with decreased affinity. In the presence of ATP, ATP can occupy the NBD1 degenerate site, but the NBDs are still separated (Fig. 6B and 6F). When the ADP concentration increases, Mg-ADP first binds to the NBD2 consensus site to induce a conformational change of NBD2 and enhance the affinity of the NBD1 degenerate site for Mg-ADP or Mg-ATP (Fig. 6C and 6G). Then, prebound ATP together with Mg ion or newly bound Mg-ADP bridges two halves of the degenerate site to induce its full closure and NBD dimerization. Finally, there is a global conformational change of SUR1, which allosterically activates the Kir6.2 channel and occludes the GBM site of SUR1, and Kir6.2 N-terminus relocates accordingly (Fig. 6D and 6H). Mg-ATP binding at the NBD2 consensus site can have similar functions as Mg-ADP, albeit with lower affinity (Vedovato et al., 2015). Our model suggests that the essential elements for asymmetric NBD dimerization are the degenerate site of both NBDs and the consensus site of NBD2. Indeed, previous mutation results support this model. Mutations of K719A and D854N on the degenerate site of NBD1; G1479D, G1479R, G1485D, G1485R and Q1486H on the degenerate site of NBD2; and K1385M, D1506N, and D1506A on the consensus site of NBD2 (Gribble et al., 1997; Nichols et al., 1996; Shyng et al., 1997) can impair Mg-ADP activation, whereas mutations of G827D, G827R, and Q834H on the consensus site of NBD1 can preserve Mg-ADP activation, although with altered kinetics (Nichols et al., 1996; Shyng et al., 1997). Our model also suggests that ATP hydrolysis is not required for channel activation, which is consistent with functional studies (Choi et al., 2008; Ortiz et al., 2013).