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• W2108706632 abstract "Article13 January 2005free access Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit Jennifer F Antcliff Jennifer F Antcliff University Laboratory of Physiology, Parks Road, Oxford, UK Search for more papers by this author Shozeb Haider Shozeb Haider Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Peter Proks Peter Proks University Laboratory of Physiology, Parks Road, Oxford, UK Search for more papers by this author Mark SP Sansom Mark SP Sansom Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Frances M Ashcroft Corresponding Author Frances M Ashcroft University Laboratory of Physiology, Parks Road, Oxford, UK Search for more papers by this author Jennifer F Antcliff Jennifer F Antcliff University Laboratory of Physiology, Parks Road, Oxford, UK Search for more papers by this author Shozeb Haider Shozeb Haider Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Peter Proks Peter Proks University Laboratory of Physiology, Parks Road, Oxford, UK Search for more papers by this author Mark SP Sansom Mark SP Sansom Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK Search for more papers by this author Frances M Ashcroft Corresponding Author Frances M Ashcroft University Laboratory of Physiology, Parks Road, Oxford, UK Search for more papers by this author Author Information Jennifer F Antcliff1,‡, Shozeb Haider2,‡, Peter Proks1,‡, Mark SP Sansom2 and Frances M Ashcroft 1 1University Laboratory of Physiology, Parks Road, Oxford, UK 2Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Oxford, UK ‡These authors contributed equally to this work *Corresponding author. Laboratory of Physiology, University of Oxford, Parks Road, Oxford OX1 3PT, UK. Tel.: +44 1865 285810; Fax: +44 1865 272469; E-mail: [email protected] The EMBO Journal (2005)24:229-239https://doi.org/10.1038/sj.emboj.7600487 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the ‘slide helix’ of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state. Introduction ATP-sensitive potassium (KATP) channels play key roles in many tissues by linking cell metabolism to electrical activity. They are involved in glucose sensing in pancreatic islets, gut L cells and the brain; in the regulation of vascular smooth muscle tone; in the response to ischaemic stress in heart and brain; and in seizure protection (Seino and Miki, 2003). KATP channels are octameric complexes of two different proteins (Clement et al, 1997). Four inwardly rectifying K+ channel subunits form a tetrameric pore: in almost all tissues except vascular smooth muscle, Kir6.2 serves this role. Each Kir subunit is associated with a regulatory sulphonylurea receptor (SUR) subunit, which belongs to the ATP-binding cassette family of transporter proteins. Metabolic regulation of KATP channel activity is mediated by changes in the intracellular concentrations of adenine nucleotides, which interact with both Kir6.2 and SUR subunits. Binding of ATP or ADP to Kir6.2 produces channel inhibition (Tucker et al, 1998; Tanabe et al, 2000), whereas interaction of Mg nucleotides with the nucleotide-binding domains (NBDs) of SUR stimulates channel activity (Nichols et al, 1996; Gribble et al, 1997). Loss-of-function mutations in both Kir6.2 and SUR1 genes cause congenital hyperinsulinism (Dunne et al, 2004). Conversely, mutations in Kir6.2 that lead to reduced ATP sensitivity of the channel cause permanent neonatal diabetes, which in some cases is associated with muscle weakness, developmental delay and epilepsy (Gloyn et al, 2004). To understand how ATP inhibits the KATP channel and how mutations in Kir6.2 impair this process, it is necessary to identify the location and structure of the ATP-binding site. The structure of this inhibitory site is unknown, but is likely to differ from that of classical ATP-binding sites, because it has several unusual properties. For example, Mg2+ is not required for the inhibitory action of the nucleotide (Ashcroft and Kakei, 1989); the site is extremely selective for the adenine base (Tucker et al, 1998), and addition of bulky groups to the end of the phosphate chain does not abolish the inhibitory effect of ATP (Ämmälä et al, 1991; Tanabe et al, 2000). Many experiments suggest that the ATP-binding site involves residues from both the N- and C-terminal domains (N and C domains) of Kir6.2 (Drain et al, 1998; Proks et al, 1999; Tucker et al, 1998; Reimann et al, 1999; Cukras et al, 2002; John et al, 2003; Ribalet et al, 2003; Tsuboi et al, 2004). In addition, although the primary site at which ATP mediates channel inhibition lies on Kir6.2, coexpression with SUR enhances the potency of ATP block about 10-fold (Tucker et al, 1997). Attempts to crystallize Kir6.2, or its C domains, have proved unsuccessful. However, the crystal structures of a putative bacterial Kir channel, KirBac1.1 (Kuo et al, 2003), and of the N and C domains of Kir3.1 (Nishida and MacKinnon, 2002), have recently been solved. We have therefore used these structures to construct a molecular model of the Kir6.2 tetramer, and ligand docking to identify residues interacting with ATP. The model is consistent with a large amount of functional data and was further tested by additional site-directed mutagenesis. Our results indicate the location and structure of the four ATP-binding sites, and suggest a possible model for how binding of ATP is transduced to transmembrane elements of the channel and thus to closure of the pore. Results Construction of a molecular model of Kir6.2 A homology model of Kir6.2 (GenBank D50581) was constructed based on the X-ray crystal structures of KirBac1.1 (Kuo et al, 2003) and the intracellular (IC) domains of Kir3.1 (Nishida and MacKinnon, 2002). KirBac1.1 was used as a template for the proximal N domain and the transmembrane (TM) domains of Kir6.2, whereas the distal N domain and the C domain were modelled on the IC domains of Kir3.1 (Figure 1A). This was because, over the region modelled, the C domains of Kir3.1 and Kir6.2 exhibit a greater sequence identity (48%) than those of Kir6.2 and KirBac1.1 (27%). Further, the crystal structure of the IC domain of Kir3.1 was determined at a resolution higher (2.0 Å) than that of KirBac1.1 (3.6 Å). Each of the three segments of the model (TMs, N and C domains) were constructed separately, and then joined together. The spatial orientation of the IC domain, with respect to the TMs, was determined from the location of conserved residues in the IC domain of KirBac1.1. Figure 1.(A) Alignment of Kir3.1, Kir6.2 and KirBac1.1 sequences. Regions boxed in red and black show residues in Kir3.1 and KirBac1.1, respectively, used to construct the model. (B, C) Model of the Kir6.2 tetramer viewed from the side (B) or above (C). (B) Residues are shown in ribbon format, with different colours representing individual subunits. (C) TMs are shown in backbone format and IC domains in ribbon format. Different colours indicate individual subunits, with dark and light shades representing the N and C domains, respectively. Download figure Download PowerPoint The structure of the N domain of Kir3.1 was resolved between residues 43 and 57 (Nishida and MacKinnon, 2002), which correspond to residues 32–46 of Kir6.2 (Figure 1A). The position of these residues with respect to the C domain of Kir6.2 is fixed by the crystal structure of the IC domain of Kir3.1. Residue 51 in Kir6.2 is also fixed, by the position of residue 63 in the Kir3.1 crystal structure (Figure 1A). The residues corresponding to positions 47–50 of Kir6.2 were not resolved in the crystal structure of Kir3.1: therefore, they were modelled as a loop and energy minimized. The minimization procedure did not significantly alter the position of the backbone of residue 50 in Kir6.2. The Cα r.m.s.d. between the N domain of Kir3.1 (template) and the N domain of Kir6.2 was 0.4 Å. The C domain of Kir6.2 was built as described previously (Trapp et al, 2003). The Cα r.m.s.d. between the Kir3.1 C-domain (template) and the Kir6.2 C-domain (model) was 1.3 Å and that between the TMs of KirBac (template) and Kir6.2 (model) was 1.7 Å. These values lie within the range expected for proteins sharing 25% sequence identity (0.7–2.3 Å) (Russel et al, 1997). The position of the ATP-binding site was predicted using ligand docking. ATP was randomly positioned on the C-domain model and 25 dockings were carried out (Trapp et al, 2003). No information about the potential binding site obtained from mutagenesis studies was specified in the docking procedure. In 19/25 runs (76%), the β-phosphate of ATP interacted with K185 (which is suggestive of an energy minimum on the potential energy landscape). The rest of the molecule lay in an identical position in five runs, and close to one or more of a consensus set of residues in all cases. In similar studies, ADP found the same set of residues as ATP 16/25 times (64%), but GTP, azido-ATP, ADP-ribose and ATP-ribose never localized to the same position. The ligand-docking program used did not allow for reorientation of side chains in response to the presence of ATP. It is clear that this would occur, however, because of the strong negative charge of the phosphate tail. Thus, the side chains of R201 and K185 were manually oriented to point towards the ATP molecule and then minimized. However, the backbone was not altered. The positions of the side chains of other residues, including R50, were not adjusted. Automated docking on the modified model revealed that the β-phosphate of ATP found K185 in all 25/25 runs. The Kir6.2–ATP complexes were then evaluated based on interaction energies between ATP and the protein, in order to determine the conformation of the docked ATP and the residues that contribute to its binding site. ATP was then superimposed in the same spatial orientation in the other subunits within the tetrameric model. It is likely that the crystal structures of both KirBac1.1 and Kir3.1 represent the closed state of the channel. First, KirBac1.1 is clearly closed, as bulky side-chain residues occlude access of hydrated K+ to the intracellular mouth of the pore (Kuo et al, 2003). Second, Kir3.1 is activated by binding Gβγ proteins, and the IC domains were crystallized in the absence of ligand. Thus, we assume that these structures form a reasonable template for the closed state of Kir6.2, in which ATP is bound. The KATP channel comprises both Kir6.2 and SUR subunits, and it is possible that SUR imposes conformational changes on Kir6.2. Thus, our model corresponds most closely to Kir6.2ΔC, a truncated form of Kir6.2 that expresses in the absence of SUR1 (Tucker et al, 1997). Thus, in our discussions, we distinguish functional data obtained for Kir6.2ΔC from that measured for Kir6.2/SUR1 channels. Overall structure of Kir6.2 Figure 1B and C give the overall structure of the tetrameric model of Kir6.2. It can be divided into two main regions, with the ‘slide helix’ (residues 54–66) lying at their interface. The TM region is 44 Å long (measured as the Cα–Cα distance between residues P109 and F60) and the cytosolic (IC) domain is 63 Å long (D65–A358). When viewed from above, the TM tetramer forms a square with dimensions of 46 Å × 46 Å between the furthest points on adjacent subunits (E104–P102). It sits on a pedestal-like IC domain, which measures 55 Å × 55 Å between D323 and D323 of adjacent subunits. The cytosolic pore that permeates the IC domain ranges from 13 to 50 Å in diameter. The TM domains. Kir6.2 has two membrane-spanning α-helices (TM1 and TM2) per subunit. TM1 and TM2 are connected via loop segments and a short α-helix (the pore helix) that loops partway into the membrane. The selectivity filter, which lies within the loop linking the pore helix to TM2, exhibits the characteristic signature sequence of K+ channels, except that GYG is replaced by GFG. The r.m.s.d. between the TMs of the present model and that of an earlier model of the TMs alone based on KcsA (Capener et al, 2003) was 2.2 Å. Most of the variation resides in the extracellular loops that connect TM1 with the pore helix (residues 92–116). If these are omitted, the r.m.s.d. between the two models is excellent: ∼0.8–1.1 Å. The IC domain. The IC domain shows extensive interactions between subunits: thus, the C domain of one subunit interacts both with the N domain of the adjacent subunit on one side, and with the C domain of the adjacent subunit on the other side (Figure 1C). The region of the N domain involved is consistent with protein–protein interaction studies, which implicate residues 30–46 as binding to the C domain (Tucker and Ashcroft, 1999). The model predicts several interactions within the IC domain of Kir6.2, which may help to stabilize the tetrameric structure. These include a π-stacking interaction between F35 in the N domain of one subunit and Y326 in the C domain of the adjacent subunit (Figure 2). Two intersubunit ion pairs, R32–E321 and R34–E308, also connect these domains (Figure 2). Several electrostatic interactions are found between the C domains of adjacent subunits (Figure 2). These include three ion pairs: E229 with R314, R301 with E292 and R192 with E227. Figure 2.Interactions between adjacent subunits. The C domain of one subunit is shown in grey and its N domain in yellow. The C domain of the adjacent subunit is shown in green. Residues are shown in cpk colours. Download figure Download PowerPoint Interactions between the IC and TM domains. There are three main sites of interaction between the IC and TM domains. The N domain is linked directly to the slide helix (Figures 1B and 3). The C domain connects directly to TM2, but also appears to be linked to the slide helix of the adjacent subunit by two loops that span residues 204–209 (loop 1) and 289–299 (loop 2) (Figure 3A and B). A web of interactions link loops 1 and 2 both to the slide helix and to the ATP-binding pocket (Figure 3C and D). Figure 3.(A, B) Relationship between the N domain of one subunit (yellow) and the C domain of another (green), showing the relation of loops 1 and 2 to the slide helix. (C) Interactions between loops 1 and 2 and the C domain of one subunit and the slide helix of the adjacent subunit. (D) Web of predicted interactions between the C linker, loops 1 and 2 of the C domain of one subunit (yellow) and the slide helix of the adjacent subunit (blue). Coloured lines connect interacting residues, or indicate interactions with ATP. Download figure Download PowerPoint As homology models of the TMs of Kirs have been constructed and evaluated previously (Loussouarn et al, 2001; Capener et al, 2003), in this paper we focus on the IC domain, and the ATP-binding site in particular. We also consider the slide helix which links the TM and IC domains, as its position suggests that it may be involved in gating of the pore, both in the absence and presence of ATP. The ATP-binding site There are four ATP-binding pockets, one per subunit, as predicted by functional studies (Markworth et al, 2000). Each is located in the upper part of the IC domain, about 17 Å below the plane of the membrane. The position of this pocket on the outer face of the protein (Figure 4A and B) may facilitate access of cytosolic ATP to its binding site. Figure 4.(A) Side view of the ATP-binding site. For clarity, the TMs of only two subunits and the IC domains of two separate subunits are illustrated. ATP (yellow) is docked into its binding sites. (B) Kir6.2 tetramer, viewed from above, with the TMs removed (residues 64–177). ATP (yellow) is docked into its binding sites. The N domain is shown in ribbon format and the C domain in backbone format. Different colours represent individual subunits. Download figure Download PowerPoint The main binding pocket lies at the interface between the N and C domains of the same subunit. In addition, the N domain of the adjacent subunit loops across the C-terminal part of the binding pocket, such that the side chain of R50 lies to one side of the entrance to the pocket, while K185 from the other subunit lies at the opposite side (Figure 5A and B). Docking of ATP was conducted in the absence of the N-terminal residues, but it is clear that the location of R50 in the complete, tetrameric, homology model would restrict access of ATP to its binding site. Thus, either the position of R50 is incorrect, or the N-domain must move to enable ATP to enter the binding pocket. Figure 5.(A) Interactions between ATP and residues lining the ATP-binding pocket. The subunit origin (A or D) of the residue is indicated. Predicted hydrogen bonds are indicated by dashed lines and hydrophobic interactions by sunbursts. Residues are shown in cpk colours. (B) Space-filling model of the ATP-binding pocket. Different subunits are indicated in blue and pink. The electron shells of R50 and K185, and of ATP, are shown in transparency. (C) Close-up of the binding pocket with 8-azido-ATP placed in the same position as ATP. The dashed lines indicate where the azido group makes a steric clash with T180 and the ATP molecule itself. Download figure Download PowerPoint The overall size of the ATP-binding pocket is sufficient to accommodate the ATP molecule (volume ∼1070 Å3). The Cα–Cα distance from A300, which forms the innermost residue of the hydrophobic pocket, to G334, which forms the outer boundary, is about 17 Å. The width of the pocket, measured from K39 to T180, is ∼10 Å and its height, measured between residues E179 and F183, is ∼8 Å. In all, 17 residues lie within 4.5 Å of ATP in the model: K38, K39, G40 in the N domain, E179, T180, L181, I182, F183, S184, K185, R201, A300, R301, T302, F333 and G334 in the C domain, and R50 from the N domain of the adjacent subunit. The adenine ring and the ribose sugar of the nucleotide are positioned on one side of a β-sheet and the phosphate tail on the other, which effectively separates the charged and uncharged groups of ATP. The adenine ring of ATP lies within a mainly hydrophobic pocket, lined by the side chains of E179, T180, L181, I182, and the backbone atoms of K38, K39, G40 and R301. The side chain of E179 and the carbonyl oxygen atom of the backbone of R301 make hydrogen bonds with the N6 atom in the adenine ring (Figure 5A). The adenine ring is accommodated tightly within the ATP-binding pocket (Figure 5B), and addition of an azido group at the 8′ position would cause a steric clash with the ribose moiety of ATP and with the side chain of T180 (Figure 5C). This explains why 8′-azido-ATP fails to dock into the ATP-binding site of the model or to inhibit the channel (Tanabe et al, 1999). No interactions are predicted with the ribose moiety of ATP, but S184 lies under the ribose and K38 alongside it. The phosphate tail of ATP makes putative electrostatic interactions with three positively charged residues: R201 with the α-phosphate, K185 with the β-phosphate and R50 (from the adjacent subunit) with the γ-phosphate (Figure 5A). Although the residue equivalent to R50 is absent in the crystal structure of the IC domains of Kir3.1, on which this model is based, the adjacent residue (equivalent to E51) is not. Thus, the position of E51 is fixed, which also restrains the predicted position of R50 and brings it close enough to interact with the γ-phosphate of ATP, as predicted by ATP-binding (Tanabe et al, 1999) and thiol modification (Trapp et al, 2003) studies. The γ-phosphate is not buried in the protein, as in many ATP-binding sites, but points out into the extracellular solution (Figure 4). Predictive power of the model The model accounts for a large amount of published data and enables previously puzzling findings to be explained (see Discussion). This provides significant validation of the model, as none of this information was used in its construction. In addition, we made new mutations to test the predictive power of the model. ATP-binding site. Of the 17 residues that lie within 4.5 Å of ATP in the model, most have already been mutated in Kir6.2ΔC or Kir6.2/SUR1. The effects of these mutations are discussed below. However, residues A300 and F333 have not been mutated in either construct, nor have L181, F183, R201, A300, R301, T302 and F333 been mutated in Kir6.2ΔC. To test the model, we examined the effects of mutating (in Kir6.ΔC) residue R201, which is predicted to make an electrostatic interaction with the α-phosphate of ATP, residues A300 and F333, which lie close to the β-phosphate of ATP, and residue E179. In addition to measuring concentration-inhibition curves for ATP, we also examined the channel open probability (Po) in the absence of ATP (which we define as the intrinsic Po) by single-channel recording. This is because mutations that increase intrinsic Po indirectly reduce KATP channel ATP sensitivity (Shyng et al, 1997; Trapp et al, 1998; Cukras et al, 2002). Thus, we only considered mutations that alter ATP sensitivity without affecting intrinsic Po as influencing ATP binding and/or transduction. The side chain of E179 is predicted to make a hydrogen bond with the N6 atom in the adenine ring of ATP (Figure 5A). To test this, we mutated E179 to a range of residues, some of which are capable of forming H bonds (Q, N), and some of which are not (M, L). Consistent with this idea, mutation of E179 to glutamine has no effect, whereas mutation to methionine reduced the IC50 two-fold (Figure 6, Table I). However, mutation to leucine caused a small but insignificant shift in IC50, which does not support an important role for H bonding. None of these mutations altered the intrinsic Po (Table I). Mutation of E179 to asparagine caused an ∼7-fold reduction in the IC50 for ATP inhibition, but also increased the intrinsic Po (Figure 6, Table I). This change in Po could account for the reduced ATP sensitivity (Shyng et al, 1997; Trapp et al, 1998; Cukras et al, 2002) and this mutation therefore cannot be used to address the role of H bonding. The relatively small effect of mutating E179 may explained by the fact that the N6 atom is also stabilized by H bonding to the backbone of R301 (Figure 5A), and further suggests that R301 may be more important in co-ordinating the adenine moiety. That E179 and ATP are slightly out of plane, and so likely to form only a weak H bond, whereas R301 and ATP lie in the same plane, is harmonious with this idea. Figure 6.(A) KATP currents elicited by voltage ramps from −110 to +100 mV to an inside-out patch excised from a Xenopus oocyte expressing Kir6.2ΔC or Kir6.2ΔC-E179M. The dotted line indicates the zero current level. (B) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc) for Kir6.2ΔC with E179 mutated to N (, n=5), M (, n=5), L (, n=5) and Q (, n=7). The curves are the best fit to equation (1), using values for IC50 and h given in Table I. The dashed line indicates the wild-type data. Download figure Download PowerPoint Table 1. Mean parameters for ATP inhibition and open probability of wild-type and mutant channels Mutation IC50(n) h Po Kir6.2ΔC (wt) 194±10 μM (n=5) 0.96±0.04 0.08±0.02 (n=6) Kir6.2ΔC-V59G 1.7±0.1 mM*** (n=5) 1.1±0.1 0.78±0.03*** (n=6) Kir6.2ΔC-E179Q 240±36 μM (n=7) 0.88±0.5 0.10±0.01 (n=7)a Kir6.2ΔC-E179L 299±47 μM (n=5) 0.89± 0.06 0.14±0.01 (n=5) Kir6.2ΔC-E179M 515±51 μM** (n=5) 1.08±0.17 0.09±0.02 (n=5) Kir6.2ΔC-E179N 1.4±0.23 mM*** (n=6) 1.3±0.1 0.58±0.03*** (n=8) Kir6.2ΔC-R201C 14±l mMb*** (n=6) 0.81±0.05b 0.12±0.02 (n=6) IC50=ATP concentration at which inhibition is half-maximal. h, Hill coefficient. Values indicate the means of the individual fits to equation (1), for n patches. aFrom Tucker et al (1998). bEstimated by fitting line through data points that extend to 10 mM. Statistical significance was determined using Student's t-test. **P<0.01; ***P<0.001. The α-phosphate of ATP is predicted to form an electrostatic interaction with the side chain of R201 (Figure 5A). Consistent with this prediction, mutation of R201 to cysteine results in permanent neonatal diabetes in humans (Gloyn et al, 2004), and causes a marked reduction in the ATP sensitivity of Kir6.2ΔC (Figure 7): the IC50 could not be measured accurately because even as much as 10 mM ATP only inhibited the channel by 43±5% (n=6), compared with 96±1% (n=5) for the wild-type channel. The IC50 was estimated to be 14 mM, 70-fold larger than the wild-type channel (Table I). Figure 7.(A) Kir6.2ΔC and Kir6.2ΔC-R201H currents evoked by voltage ramps from −110 to +100mV. (B) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc) for Kir6.2ΔC-R201C (n=5). The curve is the best fit to equation (1) using the values given in Table I. The dotted line indicates the wild-type data. Download figure Download PowerPoint We next tested the effect of mutating F333, which is predicted to lie within 3 Å of the α-phosphate of ATP (Figure 8A). In this experiment, we used full-length Kir6.2. As full-length Kir6.2 does not express by itself, it was coexpressed with SUR1: to avoid the stimulatory effects of Mg nucleotides on SUR1, we used a mutant SUR1 (SUR1-KA/KM) that is not modulated by adenine nucleotides (Gribble et al, 1997). This ensures that only ATP binding to Kir6.2 influences channel activity. The F333L mutation markedly increased the IC50 for channel inhibition—from 17 to 514 μM (Figure 8B). We also tested the effect of mutating Y330, which lies at some distance from ATP in the model, to leucine. This had no effect (Figure 8B). The model predicts that A300 lies within 4.5 Å of the adenine ring of ATP (Figure 8A). Unfortunately, mutation of A300 to either D or L failed to yield functional currents. Figure 8.(A) Model of the ATP-binding pocket of Kir6.2 illustrating the proximity of F333 and Y330 to the phosphate tail of ATP. (B) Mean relationship between [ATP] and KATP conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc) for Kir6.2-Y330L/SUR1-KA/KM (•, n=5), and Kir6.2-F333L/SUR1-KAKM (▪, n=6) channels. The smooth curves are the best fit to equation (1). For Kir6.2-Y330L, IC50=16.9±1.9 μM, h=1.3±0.1. For Kir6.2-F333L, IC50=514±93 μM, h=1.2±0.1. The dashed line indicates the control data (IC50=17 μM, h=0.99; Gribble et al, 1997). Download figure Download PowerPoint The model predicts that addition of an azido group at the 2′ position on the adenine ring will impair ATP binding, due to a steric clash with the backbone of K38 and K39. Consistent with this prediction, 2-azido-ATP blocked Kir6.2ΔC currents by 6±4% at 100 μM and by 47±2% at 1 mM (n=6). Assuming a Hill coefficient of 1, the IC50 of was 1.2 mM, ∼10-fold greater than for ATP. The ribose moiety of ATP does not make a direct interaction with the channel (Figure 5A). However, it lies within 4 Å of K185, which may explain why substitution of ribose with 2′-deoxyribose or ribose 2′,3′-dialdehyde reduces the ATP sensitivity of native skeletal muscle KATP channels (Spruce et al, 1987). At a concentration of 1 mM, 2′-deoxyribose ATP inhibited Kir6.2ΔC by 61±2% (n=11) as compared with 82±3% for ATP (n=16). Slide helix mutations The position of the slide helix suggests that it may be involved in channel gating (Kuo et al, 2003). With the exception of R54 at the N-terminal end (Cukras et al, 2002; Schulze et al, 2003), no mutations in the Kir6.2 slide helix have yet been studied: thus, we tested this idea by mutagenesis of Kir6.2ΔC. We chose to mutate V59 to glycine (V59G), because this mutation produces neonatal diabetes with developmental delay, muscle weakness and epilepsy (Gloyn et al, 2004). Figure 9 shows that the V59G mutation caused a marked increase in the intrinsic Po, which was accompanied by a 10-fold reduction in ATP-sensitivity (Table I). Figure 9.(A) Slide helix of Kir6.2, showing the location of V59. The slide helix of one subunit is shown in yellow and the TM domain of the adjacent subunit in green. (B) Kir6.2ΔC and Kir6.2ΔC-V59G currents evoked by voltage ramps from −110 to +100 mV in inside-out patches. (C) Mean relationship between [A" @default.
• W2108706632 created "2016-06-24" @default.
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• W2108706632 date "2005-01-13" @default.
• W2108706632 modified "2023-09-27" @default.
• W2108706632 title "Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit" @default.
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