Biochem. J. (2014) 464, 343–354 (Printed in Great Britain)

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doi:10.1042/BJ20140273

*Department of Cardiovascular Sciences, University of Leicester, Cardiovascular Research Centre, Glenfield General Hospital, Leicester LE3 9QP, U.K. †Department of Biochemistry, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, U.K.

ATP-sensitive potassium channels play key roles in many tissues by coupling metabolic status to membrane potential. In contrast with other potassium channels, the pore-forming Kir6 subunits must co-assemble in hetero-octameric complexes with ATP-binding cassette (ABC) family sulfonylurea receptor (SUR) subunits to facilitate cell surface expression. Binding of nucleotides and drugs to SUR regulates channel gating but how these responses are communicated within the complex has remained elusive to date. We have now identified an electrostatic interaction, forming part of a functional interface between the cytoplasmic nucleotide-binding domain-2 of SUR2 subunits and

the distal C-terminus of Kir6 polypeptides that determines channel response to nucleotide, potassium channel opener and antagonist. Mutation of participating residues disrupted physical interaction and regulation of expressed channels, properties that were restored in paired charge-swap mutants. Equivalent interactions were identified in Kir6.1- and Kir6.2-containing channels suggesting a conserved mechanism of allosteric regulation.

INTRODUCTION

respectively [13]. The presence of Mg-ADP at NBD2, either by direct binding or as a result of hydrolysis of bound MgATP, results in KATP channel activation [14], possibly due to a reduction in the affinity of Kir6 subunits for ATP [15,16]. The combined sensitivities of SUR and Kir6 subunits to nucleotides thus produce a co-ordinated regulation of channel activity in response to changing ATP:ADP ratio to reflect cellular energy status. In addition to opening in response to ADP and closing in response to ATP, channel gating can be stimulated by potassium channel opening (KCO) drugs, such as nicorandil and inhibited by the anti-diabetic sulfonylureas, such as glibenclamide. Binding sites for channel openers and antagonists have been defined in SUR subunits [17,18] but how binding to SUR is converted into a response by the pore is unknown. Likewise, although the interplay between nucleotide regulation of KATP channel activity through the two subunits has been investigated in some detail [14], the molecular interactions underpinning this allosteric regulation remain unknown. In pursuit of a hypothesis that regulatory conformational change is transmitted between cytoplasmic structures in the two subunits, we identified a sequence (residues 1294–1358) in NBD2 of SUR2 that bound Kir6.2 [19]. Disruption of this interaction with peptide mimetics of the SUR2 NBD2 sequence inhibited the cell surface expression of KATP channels, indicating an important role in stabilizing channel assembly [20]. We have now identified contributory residues within the interaction site on SUR2 NBD2 (Glu1318 , Lys1322 and Gln1336 ) and the corresponding binding region in the C-terminal region of Kir6.2 (residues 316–390). Our results identify three intersubunit electrostatic interactions between these domains (Kir6.2-Lys338 –SUR2A-Glu1318 , Kir6.2-Asp323 –SUR2ALys1322 and Kir6.2-Asp323 –SUR2A-Gln1336 ) and we show that the

The ATP-sensitive potassium (KATP ) channel is a widely expressed complex intimately involved in a number of important physiological processes. In addition to well-characterized roles in pancreatic insulin secretion and regulation of vascular tone, the channel is also abundantly expressed in the myocardium and neural tissue and has been implicated in cardioprotective and neuroprotective processes respectively. Mutations causing gain or loss of function have been shown to have devastating consequences for normal physiological function; permanent neonatal diabetes, developmental delay with epilepsy, muscle weakness and neonatal diabetes (DEND) syndrome and congenital hyperinsulinism [1,2]. KATP channels include a potassium-selective pore, formed from four inward rectifier potassium channel Kir6 subunits [3–5], which is closed by the binding of ATP [6]. Cell surface expression of these channel subunits requires co-expression of four accessory sulfonylurea receptor (SUR) subunits that provide complex regulation of channel activity in response to nucleotide binding [7,8]. This makes the channel unique in that it is dependent on its β-subunit for both trafficking and gating. This dependence is reflected in a complex channel biosynthesis including multiple mechanisms to prevent the expression of incomplete (and therefore disregulated) assemblies at the membrane [9]. SUR subunits are members of the ATP-binding cassette (ABC) superfamily of regulatory proteins [10]. They are composed of three transmembrane helical bundles, transmembrane domain (TMD) 0, TMD1 and TMD2, connected by long cytoplasmic linker sequences [11,12]. The defining structures are nucleotidebinding domains-1 and -2 (NBD1 and NBD2), located between TMD1 and TMD2 and in the cytoplasmic C-terminal domain

Key words: allosteric regulation, glibenclamide, inward rectifier, KATP channel, pinacidil, sulfonylurea receptor.

Abbreviations: ABC, ATP-binding cassette; 2CaT, 2 mM Ca2 + Tyrode’s solution; DEND, developmental delay with epilepsy, muscle weakness and neonatal diabetes; HEK, human embryonic kidney; KATP , ATP-sensitive potassium (channel); KCO, potassium channel opening drug; MBP, maltosebinding protein; MRP1, multidrug-resistance-associated protein 1; NBD, nucleotide-binding domain; PC, proximal C-terminal; SUR, sulfonylurea receptor; MI, metabolic inhibition; TMD, transmembrane domain; WT, wild-type. 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed (email [email protected]).  c The Authors Journal compilation  c 2014 Biochemical Society

Biochemical Journal

David Lodwick*1,2 , Richard D. Rainbow*1 , Hussein N. Rubaiy*, Mohammed Al Johi*, Geerten W. Vuister† and Robert I. Norman*

www.biochemj.org

Sulfonylurea receptors regulate the channel pore in ATP-sensitive potassium channels via an intersubunit salt bridge

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Figure 1

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Co-immunoprecipitation of MBP–SUR2A–MRP1-PC chimaeric fragments with Kir6.2 subunit

(a) Schematic representation of chimaeric SUR2A–MRP1 NBD2 PC constructs showing residue boundaries. Residue numbers are shown under each bar. MRP1, white bars; SUR2A, black bars; Walker-A motif; grey box. MRP1-PC-(1280–1389); SUR2-PC-(1294–1403); SMM, SUR2-(1294–1317)–MRP1-(1306–1389); SSM, SUR2-(1294–1337)–MRP1-(1324–1389); MSS, MRP1-(1280–1305)–SUR2-(1318–1403); MMS, MRP1-(1280–1324)–SUR2-(1338–1403). (b) Immunodetection on Western blots of co-immunoprecipitated MBP–SUR2A–MRP1-PC fusion proteins with WT Kir6.2 using Kir6.2 antiserum. Immunoprecipitation experiments were performed in the presence (lane 1) or absence (lane 2) of Kir6.2. Lane 3, direct load of 12 ng of MBP–SUR2–MRP1-PC fusion protein. (c) Mean + − S.E.M. co-immunoprecipitation determined by densitometry (n = 3).

first of these forms a salt bridge, regulating transmission of regulatory information to the channel pore in response to the binding of a potassium channel opener (pinacidil), an antagonist (glibenclamide) and metabolic regulator (ADP) to the SUR2 subunit.

chromatography on amylose–Sepharose (New England Biolabs). Purity was verified by SDS/PAGE (7.5 % gels) with Coomassie Blue staining. Co-immunoprecipitation of MBP–rSUR2A-CT protein fragments

EXPERIMENTAL Molecular biology

Point mutant and chimaeric constructs were produced by overlap PCR with inserts confirmed by DNA sequencing. For electrophysiology, Kir6.0 wild-type (WT) and mutants were expressed in pcDNA3.1/myc/hisA (Invitrogen) and SUR2A WT and mutants were expressed in pIRES2-EGFP-F [19]. SUR protein fragments

Fragments of rat SUR2A (Figure 1a) (amino acids 1294–1403), the equivalent region of rat multidrug-resistance-associated protein 1 (MRP1) (amino acids 1280–1389) and chimaeric constructs and point mutants thereof were expressed as maltosebinding protein (MBP) fusion proteins using the pMAL protein fusion system (New England Biolabs). Proteins were expressed in Escherichia coli BL21(DE3) cells and purified by affinity  c The Authors Journal compilation  c 2014 Biochemical Society

MBP-labelled NBD2 sequence fragments of SUR2A, MRP1 or SUR2A–MRP1 chimaeric fragments were coimmunoprecipitated with in vitro translated unlabelled or [35 S]methionine-labelled Kir6.2, Kir2.1 or Kir6.2–Kir2.1 chimaeric subunits, as required, using methods essentially as described previously [19]. Chimaeras of Kir6.2–Kir2.1 [21,22] were gifts from Professor Andrew Tinker (University College London, London, U.K.) and Professor Asipu Sivaprasadarao (School of Biomedical Sciences, University of Leeds, Leeds, U.K.). N- and C-terminal epitope tags on the original constructs were removed before experimentation. Co-immunoprecipitation reactions using either previously characterized anti-Kir6.2 [23] or anti-Kir2.1 [24] antisera were carried out as described previously [19]. Where required, immunoprecipitated 35 S-labelled Kir subunit was visualized by autoradiography on X-ray film (Kodak) and quantified by densitometry, to permit normalization of MBP-labelled NBD2 fragment immunoprecipitation to 35 S-labelled Kir subunit in the precipitate.

Allosteric regulation of KATP channels Tissue culture

Human embryonic kidney (HEK)-293 cells were transiently transfected with both Kir6.2 and SUR2A subunits, or mutant subunits as indicated, 48 h before patch-clamp investigations. Cells were released from the plate by a 2 min trypsin digestion (0.5 g/l trypsin, 0.2 g/l EDTA; Sigma), followed by repeated washing with sterile 2 mM Ca2 + Tyrode’s solution (2CaT) (6 mM KCl, 135 mM NaCl, 0.33 mM NaH2 PO4 , 5 mM sodium pyruvate, 10 mM glucose, 10 mM Hepes, 2 mM CaCl2 and 1 mM MgCl2 ), adjusted to pH 7.4 with NaOH. Cells were maintained in 2CaT at room temperature until use. For excised patch recording, cells were cultured and transfected on coverslips 48 h before use. Patches were excised from the cells already adhered to the coverslip. Homology modelling of the distal Kir6.2 C-terminal–SUR2 NBD2 interaction

Homology modelling of the distal Kir6.2 C-terminal (rabbit residues 1–390) and SUR2A NBD2 (mouse residues 1076– 1545) was conducted using the program YASARA Structure (http://www.yasara.org) and yielded Kir2.2 (PDB code 3SPC, quality score 0.462; cover 84 %) and Abcb10 human mitochondrial ABC transporter (PDP code 4AYX, quality score 0.578, cover 91 %) as the highest scoring homology templates respectively. For Kir6.2, template matching yielded 80.3 % alignment with 52.1 % identity and 71.9 % similarity and the final tetrameric model displayed the expected four-fold symmetry with residues 31–357 reliably included in the model. For SUR2A, template matching yielded 91.9 % alignment with 25.5 % identity and 45.1 % similarity and the final model was a symmetric dimer with two copies of the modelled segment, of which only one was subsequently considered. A model of the heterologous subunit complex was generated by orienting both the Kir6.2 and SUR2A such that their respective TMDs aligned and their symmetry-axes were approximately perpendicular to the hypothetical membrane surface, in line with the arrangement of the Kir6.2–SUR2A complex derived from EM data [25]. Given the biochemical data indicating the Kir6.2Lys338 –SUR2A-Glu1318 interaction, the close proximity of Kir6.2-Lys338 and SUR2A-Glu1318 was imposed as a restricting requirement during the docking procedure. Three successive rounds of manual docking and side-chain optimization, followed by short simulated annealing minimizations of the interface residues (Kir6.2 Thr336 –Thr345 , Ala271 –His276 and Glu308 – Pro317 ; SUR2A Cys1314 –Lys1326 and Leu1361 –Asp1363 ) using the YAMBER3 force field, yielded a model for the complex. Electrophysiology

For conventional whole cell recording from HEK-293 cells, 2CaT was used as an extracellular solution. The pipette solution (intracellular solution) contained 140 mM K + (30 mM KOH and 110 mM KCl), 10 mM EGTA, 10 mM Hepes, 0.1 mM MgCl2 , 1 mM ATP, 0.1 mM ADP and 0.1 mM GTP, adjusted to pH 7.2 with KOH. For excised inside-out patch recording, the bath and perfusing solution contained, 140 mM K + (30 mM KOH and 110 mM KCl), 10 mM EGTA, 5 mM Hepes, 1 mM CaCl2 and 1.2 mM MgCl2 , adjusted to pH 7.2 with HCl. The pipette solution contained 140 mM KCl, 5 mM Hepes, 1.2 mM MgCl2 and 2.6 mM CaCl2 , adjusted to pH 7.4 with KOH. For recordings of Kir6.1–SUR2A the pipette solution was modified, in particular to include 10 mM UDP, shown to be necessary to permit currents to be recorded from this subunit combination. Pipette solution

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contained 140 mM KCl, 10 mM EGTA, 10 mM Hepes, 2 mM MgCl2 , 1 mM CaCl2 , 0.1 mM NaADP, 0.1 mM GTP, 10 mM UDP and 1 mM ATP, adjusted to pH 7.2 with KOH (modified from [26]). Conventional whole cell recordings from HEK-293 cells were made using thick-walled filamented borosilicate glass pulled to a resistance of 3–6 M. Cells were continuously perfused at ◦ −1 32 + − 2 C at a rate of 5 ml min with 2CaT solution, with pinacidil and glibenclamide added to the solutions as indicated. Currents were recorded using an Axopatch 200B amplifier, digitized using a Digidata 1440 and recorded to computer using pCLAMP10. Data were analysed using pCLAMP10, Excel 2010 and Graphpad Prism 6. Transfected cells were identified by EGFPF fluorescence at 488 nm under mercury lamp illumination. Transfected cells were voltage-clamped at 0 mV (approximate EK − 88 mV) so a large outward current was recorded. For excised inside-out patch recording, experiments were performed at room temperature, and excised patches were placed into a perfusion tip where solutions could be perfused directly on to the patch to measure ATP and ADP sensitivity using a gravity-fed system. Excised patches were held at 0 mV (approximate EK ) and pulsed to − 80 mV for 400 ms to record the inward current in the absence of any rectification. For each concentration of ATP and ADP tested, 20 steps to − 80 mV were recorded and an average current over the last 50 ms of each sweep was used to get a mean current for the test solution for that patch. These data were then used to calculate a mean current for each solution tested over a number of patches and construct a concentration–response curve. An IC90 concentration of ATP was calculated and used to measure the ability of ADP to overcome the ATP block of the channel. RESULTS Mapping interaction between SUR2 NBD2 and Kir6.2

We have previously identified a region of SUR2 NBD2 (residues 1294–1358) that interacts with Kir6.2 [19]. To refine the interacting sequence and to define the key residues, we carried out co-immunoprecipitation experiments to establish which parts of this proximal C-terminal region were essential for interaction. Chimaeric MBP tagged constructs of SUR2 and non-interacting MRP1 were produced, such that the identified sequence (residues 1294–1358) was divided into three (Figure 1a). Co-immunoprecipitation with Kir6.2 occurred only when SUR2 residues 1318–1337 were present in the chimaera, indicating that this 20-residue-long segment was critical for interaction (Figures 1b and 1c); however, variability in the relatively small differences in signal meant that this result did not reach statistical significance. Our previous study showed that coexpression of the SUR2 NBD (residues 1294–1358) sequence fragment disrupted surface expression of WT Kir6.2–SUR2A channels. Likewise in the present study, pinacidil activated, glibenclamide-sensitive current of co-expressed WT Kir6.2– SUR2A channels was reduced significantly only by SUR2A– MRP1 NBD2 chimaeric fragments containing SUR2 residues 1318–1337, thus corroborating the co-immunoprecipitation experiments and confirming the importance of this short sequence for interaction [SUR2-(1294–1337)–MRP1-(1324–1389) and MRP1-(1280–1305)–SUR2-(1318–1403); Figures 2a and 2b]. A similar approach was used to identify the corresponding interaction domain on the Kir6.2 channel subunit. Full-length chimaeras of Kir6.2 and the non-interacting, but structurally similar, Kir2.1 were co-immunoprecipitated with MBP-tagged SUR2-(1294–1403) (Figures 3a–3c). The MBP–SUR2-(1294– 1403) fragment was immunoprecipitated only when the  c The Authors Journal compilation  c 2014 Biochemical Society

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Figure 2

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Suppression of Kir6.2–SUR2A currents by SUR2A–MRP1-PC chimaeric fragments

(a) Example recordings of currents from HEK-293 cells, stably expressing Kir6.2–SUR2A, transiently transfected with SUR2A–MRP1-PC fusion constructs (residue numbers as described in Figure 1). Currents were activated by perfusion of 100 μM pinacidil at a holding potential of 0 mV eliciting an outward current in the K + gradients used. (b) Mean + − S.E.M. whole cell current, recorded over 10 s, 3 min after starting perfusion with pinacidil, normalized to cell capacitance. (n = 4, *P < 0.05, **P < 0.01, ANOVA with Bonferroni’s post-hoc test).

C-terminal 75 residues of Kir6.2-(316–390) were present in the Kir6.2–Kir2.1 subunit chimaera (Figures 3b and 3c). The low signals for immunoprecipitation of ChmE and MBP–MRP1proximal C-terminal (PC) were regarded as non-specific trapping of the MBP fragments in the matrix used for pull-down; similar signals were seen in some control experiments (in which no Kir6.2 subunit was present). Further washing did not improve this artefact but resulted in progressive loss of specific signal.

Electrostatic interactions between SUR2 NBD2 and the distal Kir6.2 C-terminal

Mutagenesis at positions within the 20-residue SUR NDB2 sequence (Figure 4a) that are conserved in SUR1 and SUR2 subunits revealed two charged and one polar residue in SUR2, Glu1318 , Lys1322 and Gln1336 , which when changed individually to the equivalent residue in non-interacting MRP1, reduced co-immunoprecipitation with full-length Kir6.2 (all P < 0.01) (Figures 4b and 4c). To further refine the Kir6 side of the interaction, charged residues present in both SUR-interacting Kir6.2 and Kir6.1 subunits but of opposite or no charge at the equivalent positions in the non-interacting Kir2.1 subunit (Figure 5a) were selected and changed individually to the corresponding residue in Kir2.1. In addition, the Kir6.2 endoplasmic reticulum retention sequence, R369 KR371 , was replaced with AAA in one mutant. Coimmunoprecipitation of the MBP–SUR2-(1294–1358) fragment was reduced with two Kir6.2 mutants, Kir6.2-D323K (∼50 %, P < 0.05) and Kir6.2-K338E (∼80 %, P < 0.001), but not Kir6.2RKR369–371AAA or Kir6.2-K377A, thereby localizing the NBD2 interaction site within the distal Kir6.2 C-terminal domain (Figures 5d–5f). Assuming electrostatic interactions in the identified cytoplasmic contact between heterologous subunits and given the distribution of conserved charged and polar residues in the two sequences (Figures 4a and 5a), the following residue pairs were hypothesized, Kir6.2-Lys338 –SUR2-Glu1318 , Kir6.2-Asp323 –SUR2-Lys1322 and Kir6.2-Asp323 –SUR2-Gln1336 (Figure 5c). Single charge reversals in both full-length Kir6.2 and MBP–SUR2-(1294–1358) restored co-immunoprecipitation  c The Authors Journal compilation  c 2014 Biochemical Society

to at least WT levels from mixtures of Kir6.2-K338E–SUR2E1318R, Kir6.2–D323K–SUR2-K1322D and Kir6.2-D323K– SUR2-Q1336E (Figures 5e and 5f), indicating that all three of the proposed electrostatic interactions may be important structurally and/or for assembly.

Structural aspects of the distal Kir6.2 C-terminus–SUR2 NBD2 interaction

Using a homology model of the Kir6.2–SUR2A complex (Figure 6, see the Experimental section) we explored potential structural aspects of the interaction between the two subunits. All of the identified five residues are solvent exposed in the models of their respective proteins. The crucial SUR2A residues (Figure 6a), Glu1318 –Lys1322 , are part of an eight-residue loop connecting strands β1 and β2, with their side chains exposed and Gln1336 is located at the start of strand β3. Kir6.2-Lys338 is located at the tip of strand β12, its side chain facing the solvent (Figures 6a and 6d). The acid segment Glu321 –Asp323 is located at the end of strand β10 and a β-hairpin connecting it to strand β11. The model indicates that Kir6.2 and SUR2A can form a complex without serious steric clashes with Kir6.2-Lys338 –SUR2A-Glu1318 engaging in a salt-bridge [∼3.2 Å (1 Å = 0.1 nm) distance between the Lys338 Nζ and Glu1318 Cδ ] (Figure 6d). Moreover, the model shows the transmembrane regions of the two proteins sufficiently aligned for both to be immerged into the membrane. The interface between the two proteins is relatively modest, but additional hydrophobic contacts contribute favourably to the interaction. Most notably, in the model the side chain of the crucial Kir6.2Lys338 residue packs against SUR2A-Phe1362 , the hydrophobic Kir6.2-Pro340 –Pro342 loop engages with a hydrophobic pocket of the SUR2A protein formed by residues Phe1362 , His1327 , Cys1314 and the side chains of Lys1326 and Asp1363 . Moreover, Kir6.2-Trp311 is juxtaposed to SUR2A-Leu1321 . The model is less able to reconcile the interaction data between the acidic Kir6.2-Glu321 –Asp323 residues and SUR2A-Lys1322 . In the model, the Kir6.2-Asp323 residue of the same monomer as Lys338 is separated by ∼48 Å from the SUR2A-Lys1322 residue. A shorter distance, ∼29 Å, is found between Lys1322 and Asp323 of the adjacent Kir6.2 monomer. Homology modelling using

Allosteric regulation of KATP channels

Figure 3

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Co-immunoprecipitation of MBP–SUR2A-PC fragments with Kir6.2–Kir2.1 subunit chimaeras

(a) Schematic representation of Kir6.2–Kir2.1 subunit chimaeras showing amino acid boundaries. Kir6.2 sequence, thick bars; Kir2.1 sequence, thin bars. (b) Immunodetection of co-immunoprecipitated MBP–SUR2-PC (i) or MBP–MRP1-PC (ii) fusion proteins with WT Kir6.2, WT Kir2.1 or Kir6.2–Kir2.1 subunit chimaeras using Kir6.2 antiserum against the subunit C-terminal. Lanes labelled anti-Kir2.1 and anti-Kir6.2 were immunoprecipitations performed in the absence of Kir subunit protein. Direct load, 12 ng of MBP–SUR2-PC (i) and MBP–MRP1-PC (ii) fusion protein. (c) Mean + − S.E.M. co-immunoprecipitation determined by densitometry (n = 3, ***P < 0.0001, ANOVA with Bonferroni’s post-hoc test). MBP–SUR2-PC, black bars; MBP–MRP1-PC, grey bars.

Figure 4

Co-immunoprecipitation of MBP–SUR2-PC fragments containing single amino acid substitutions with Kir6.2 subunit

(a) Sequence alignments of the minimally interacting SUR2 sequence (residues 1318–1337) identified in Figure 1 with equivalent sequences of rat SUR1, MRP1 and CFTR (cystic fibrosis transmembrane conductance regulator) proteins. Acidic and basic residues are underlined in bold and grey respectively. (b) Immunodetection on Western blots of co-immunoprecipitated MBP–SUR2-PC fragments containing single amino acid substitutions with WT Kir6.2 using Kir6.2 antiserum. Immunoprecipitation experiments were performed in the presence (lane 1) or absence (lane 2) of Kir6.2. Lane 3, direct load of relevant MBP–SUR2 protein fragment. (c) Mean + − S.E.M. co-immunoprecipitation determined by densitometry (n = 5, *P < 0.05, **P < 0.01, ANOVA with Bonferroni’s post-hoc test).

 c The Authors Journal compilation  c 2014 Biochemical Society

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Figure 5 Targeted single charge reversal in both MBP–SUR2A-PC single substitution mutant fragments and full-length Kir6.2 subunit single substitution mutants restored co-immunoprecipitation (a) Sequence alignments of the C-termini of Kir6.2, Kir6.1 and Kir2.1. Acidic and basic residues are underlined in bold and grey respectively. Numbers indicate charged residues present in both SUR-interacting Kir6.2 and Kir6.1 subunits but of opposite or no charge in the equivalent positions in non-interacting Kir2.1 subunit, changed individually to the corresponding residue in Kir2.1 for further study. (b) Cartoon showing a Kir6 and SUR subunit with the putative interaction motifs in the C-terminal of Kir6 and NBF2 of TMD2 of SUR highlighted. (c) Primary sequences of the minimal interaction motifs (Kir6.2 top, SUR2 bottom) showing hypothesized cytoplasmic electrostatic interactions between heterologous subunits. (d) Immunodetection on Western blots of co-immunoprecipitated MBP–SUR2-PC WT by full-length WT and substitution mutant Kir6.2 subunits (upper panel) and amount of 35 S-labelled Kir6.2 subunit in the immunoprecipitate (lower panel). Lane 1, direct load, then paired lanes with and without Kir6.2 as indicated; lanes 2 and 3, WT; lanes 4 and 5, D323K; lanes 6 and 7, K338E; lanes 8 and 9, RKR369–371AAA; lanes 10 and 11, K377A. (e) Immunodetection on Western blots of co-immunoprecipitated MBP–SUR2-PC WT and substitution mutants by full-length WT and substitution mutant Kir6.2 subunit (upper panel) and amount of 35 S-labelled Kir6.2 subunit in the immunoprecipitate (lower panel). Lane 1, direct load, then paired lanes with and without Kir6.2 as indicated; lanes 2 and 3, WT; lanes 4 and 5, D323K; lanes 6 and 7, K338E; lanes 8 and 9, K338E and E1318R; lanes 10 and 11, D323K and K1322D; lanes 12 and 13, D323K and Q1336E. (f) Mean + − S.E.M. co-immunoprecipitation determined by densitometry and normalized to the amount of 35 S-labelled Kir6.2 subunit in the immunoprecipitate. Results were expressed in comparison with that obtained with the WT (positive control) Kir6.2 subunit. A single charge reversal point mutation, Kir6.2-D323K or -K338E, significantly reduced the co-immunoprecipitation and reinstatement of the intersubunit salt bridges by co-expression of the charge reversal residues in both subunits, Kir6.2-K338E–SUR2-E1318R, Kir6.2-D323K–SUR2A-K1322D and Kir6.2-D323K–SUR2A-Q1336E restored the co-immunoprecipitation (n = 5, *P < 0.05, ***P < 0.001, ANOVA with Bonferroni’s post-hoc test).

two additional templates indicated conformational uncertainty in the SUR2A-Glu1318 –Lys1322 loop, suggesting a potential ∼10– 15 Å variability between the Glu1318 carboxy and Lys1322 amine moieties. Even taking this variability into account, the model cannot fully reconcile all of the interaction data. Instead, SUR2A-Lys1322 appears well positioned to interact with Kir6.2Ser273 /Asp274 . It should be noted that the template restricted the  c The Authors Journal compilation  c 2014 Biochemical Society

SUR2A model to only contain residues from the C-terminal third of the full molecule and therefore lacks any structural constraints from the N-terminal part of the molecule that may be important in the native structure. Alternatively, given the presence of additional copies of the SUR2A moiety in the 4:4 overall complex [25], indirect interaction between the heterologous subunits could also be at play. A further consideration is that Kir6.2 residues

Allosteric regulation of KATP channels

Figure 6

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Homology model of the Kir6.2–SUR2A complex

(a) Overall arrangement of Kir6.2, shown as ribbons (green, orange, yellow and grey for each monomer) and SUR2A in cyan transparent surface representation and cyan ribbon. Kir6.2 residues Lys338 and Asp323 are shown in space-filling representation. (b) Ribbon diagram of SUR2A with crucial residues in stick representation. Residues 1078–1304 and 1372–1464 are omitted for clarity. (c) Detailed view of (b) with ATP bound to NBD2 and transparent surface representation of Ile1310 , Leu1313 and Leu1351 . (d) Kir6.2–SUR2A interface around the Lys338 –Glu1318 salt bridge. Colours and representation as in (a) with SUR2A Glu1318 (red) and hydrophobic surface (blue) and the Kir6.2 Pro340 –Pro342 loop (yellow).

316–323 have been identified previously as a binding site for the adapter protein ankyrin B [27] raising the possibility of varying intersubunit interactions during channel biogenesis.

Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge formation decreases sensitivity to activation by pinacidil

KATP channel opening is subject to complex regulation by nucleotides and pharmacological agents. Channel pores are inhibited directly by the binding of ATP to nucleotide-binding sites formed between the cytoplasmic N- and C- terminal sequences of Kir6 subunits. As the ATP/ADP ratio falls, KATP channel activation results from a combination of the reduction in binding of inhibitory ATP to Kir6 and an increase in stimulatory nucleotide binding to sites formed by NBD1 and NBD2 in the SUR subunits, in particular the binding of Mg-ADP to NBD2. This nucleotide binding to SUR subunits is thought to signal a reduction in the affinity of inhibitory ATP at the Kir6 subunits, thereby increasing channel opening. In addition to this nucleotide regulation, KATP channels may be activated by potassium channel openers and inhibited by antagonists acting at distinct sites on the SUR subunit. The proximity of the interacting residues to nucleotide-binding sites in each subunit was suggestive of a possible regulatory contact between cytoplasmic domains of heterologous subunits. The putative salt bridge between Kir6.2-Lys338 and SUR2Glu1318 was selected for further investigation of the functional significance of this intersubunit electrostatic interaction. Single residue replacements of either Kir6.2-Lys338 or SUR2A-Glu1318 with those found in the equivalent position in non-interacting Kir2.1 (Glu346 ) or MRP1 (Arg1306 ) respectively were made in fulllength subunits and the sensitivity of WT and mutant subunit

combinations to the potassium channel opener, pinacidil and antagonist, glibenclamide, was investigated in HEK-293 cells. In addition, sensitivity to activation by Mg-ADP and inhibition by ATP was investigated in different subunit combinations by inside-out patch recording. Sensitivity to activation by pinacidil was increased significantly when the putative Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge was disrupted by a single charge reversing mutation at position Lys338 (Kir6.2–K338E) and expressed with WT SUR2A subunits (Figures 7a and 7b, and Supplementary Table S1). The EC50 was decreased from 39.6 + − 1.3 μM in WT to 4.5 + − 0.3 μM in Kir6.2-K338E–SUR2A-E1318R channels (Figure 7b). There was no accompanying change in the peak pinacidil-activated current or the maximal current elicited by metabolic inhibition (MI) (Supplementary Table S2), indicating that functional channel pore assembly was unaffected. When the salt bridge was disrupted by a single charge mutation SUR2A-E1318R expressed with WT Kir6.2, very low pinacidil-activated current amplitude prevented an accurate determination of EC50 , although MI-activated current was similar to WT (Supplementary Table S2). This suggested a disruption of pinacidil activation through SUR2, without a gross change in expression of the channel complex. When the salt bridge was reinstated by co-expression of targeted charge reversals in both subunits (Kir6.2-K338E–SUR2A-E1318R), near WT pinacidil sensitivity (46.7 + − 4.9 μM) was measured, indicating a restoration of allostery between subunits (Figure 7b, and Supplementary Table S1); although channel density, given by pinacidiland MI-activated maximal current was approximately half that of WT (Supplementary Table S2). Together, these results suggest that salt bridge formation between Lys338 in the Kir6.2 C-terminal domain and Glu1318 in the SUR2 NBD2 stabilizes intersubunit interaction and reduces the sensitivity of channel opening in response to potassium channel opener binding to SUR2A.  c The Authors Journal compilation  c 2014 Biochemical Society

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Figure 7

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Kir6.2-K338E–SUR2A-E1318R double charge swap restores WT channel pharmacology

(a) Example recording of WT Kir6.2–SUR2A channel activation by pinacidil and inhibition with glibenclamide. (b) Mean concentration response data to pinacidil stimulation (normalized to cell capacitance) with subunits as identified in the Figure. Kir6.2-K338E and SUR2A WT formed channels which had a marked increase in sensitivity to pinacidil (EC50 = 4.5 + − 0.3 μM compared + with 39.6 + − 13 μM for WT channels, P < 0.001, ANOVA with Bonferroni’s post-hoc test). This was reversed if Kir6.2-K338E was co-expressed with SUR2A-E1318R (EC50 = 46.7 − 4.9 μM P > 0.05, ANOVA with Bonferroni’s post-hoc test, n  6 cells for each data point). Kir6.2 WT with SUR2A–E1318R formed channels that gave compared with 39.6 + 13 μM for WT channels, − very small currents with pinacidil, although a substantial current could be evoked using metabolic inhibition (Supplementary Table S2), and so a concentration–response curve could not be constructed. Pinacidil at 100 μM was found to activate this current maximally. (c) Example of glibenclamide concentration inhibition in currents recorded from Kir6.2-K338E and SUR2A WT containing channels. (d) Concentration–inhibition curves for glibenclamide for the subunit combinations shown in the Figure activated by an approximate EC50 concentration of pinacidil (100 μM pinacidil for Kir6.2 WT–SUR2A-E1318R). Both the Kir6.2-K338E–SUR2A WT and Kir6.2 WT–SUR2A-E1318R combinations showed a significant rightwards shift in IC50 (103 + − 28 nM, P < 0.05 and + 980 + − 58 nM, P < 0.001 respectively compared with 3.1 − 0.8 nM in WT, ANOVA with Bonferroni’s post-hoc test). This was attenuated by co-expression of the charge swap mutants Kir6.2-K338E + and SUR2A-E1318R (6.4 + − 0.9 nM compared with 3.1 − 0.8 nM in WT, P > 0.05, ANOVA with Bonferroni’s post-hoc test, n  6 cells for each data point).

Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge formation increases sensitivity to inhibition by glibenclamide

In contrast with the increase in sensitivity to potassium channel opener on disruption of the Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge, a single charge reversal in either subunit was sufficient to significantly reduce the sensitivity of pinacidil-activated current to inhibition by the KATP channel antagonist glibenclamide (Figures 7c and 7d, and Supplementary Table S3). Glibenclamide sensitivity was restored to near WT when the salt bridge was reinstated with a single charge reversal of target residues in both subunits (Kir6.2-K338E–SUR2A-E1318R) (Figure 7d, and Supplementary Table S3). This result implicates this salt bridge in the stabilization of conformations that define glibenclamide sensitivity in the native channel complex. ATP sensitivity of Kir6 subunits is not influenced directly by the Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge

In addition to the regulatory influence of SUR subunits on channel activity in response to binding of pharmacological agents, KATP channel pores are subject to direct inhibition by ATP through binding to Kir6 subunits and indirect regulation by nucleotide binding to SUR subunits. To ensure that the increased pinacidil sensitivity of mutants was not an indirect effect of reduced ATP sensitivity, we measured the concentration-dependence of ATP inhibition of recombinant WT and mutant subunit combinations in excised, inside-out patches. Although pinacidil and glibenclamide sensitivities were markedly altered (see above), disruption of  c The Authors Journal compilation  c 2014 Biochemical Society

the Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge by single charge reversal of Kir6.2-K338E did not change the IC50 for ATP compared with WT (Figures 8a–8c, and Supplementary Table S4) indicating that mutation of Kir6.2 close to the ATP-binding site did not alter ATP affinity directly. For comparison, mutation of Kir6.2F333I and -G334D in the same region was reported to reduce ATP sensitivity, whereas allosteric interaction with SUR2A was affected in Kir6.2-F333I–SUR2A but not Kir6.2-G334D–SUR2A [28]. A significant increase in IC50 was observed when the salt bridge was disrupted in the WT Kir6.2–SUR2A-E1318R subunit combination (Figures 8b and 8c, and Supplementary Table S4). Given the absence of change in IC50 on single point mutation of Kir6.2-K338E, it was inferred that conformational change in SUR2 was responsible for reduced ATP sensitivity in the Kir6.2K338–SUR2A-E1318R channel. Restoration of a near WT IC50 for ATP inhibition on reinstatement of the salt bridge in the Kir6.2K338E–SUR2A-E1318R subunit combination (Figures 8b and 8c, and Supplementary Table S4), indicated that this electrostatic restraint was sufficient to constrain the structure of the mutated SUR2A NBD2 and restore near native communication between the subunits.

Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge formation is not essential for allosteric coupling of ADP binding to SUR2A NBD2

ADP sensitivity of expressed WT and mutant recombinant channels was measured in excised, inside-out patches from HEK293 cells. In the absence of nucleotides, a large spontaneous

Allosteric regulation of KATP channels

Figure 8

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Altered ATP and ADP sensitivity in channels expressing the SUR2A-E1318R mutant are restored to WT when co-expressed with Kir6.2-K338E

(a) Example recording of currents from an excised inside out patch from a HEK-293 cell expressing WT Kir6.2–SUR2A showing ATP sensitivity. (b) Mean concentration–inhibition curves for the Kir6.2 and SUR2A WT and mutants as identified in the Figure. There was a significant rightward shift in the curve for the Kir6.2 WT–SUR2A-E1318R mutant which was restored to WT when co-expressed with Kir6.2-K338E mutant. (c) Mean + − S.E.M. EC50 values for the four KATP channel subunit combinations as identified in the text. These data show a significant shift in IC50 for the + + Kir6.2 WT–SUR2A-E1318R mutant that is restored on co-expression with Kir6.2-K338E (65.8 + − 4.7 μM and 27.7 − 1.5 μM respectively compared with WT, 23.8 − 1.7 μM, ***P < 0.001). (d) Relief of KATP channel inhibition, using an IC90 concentration of ATP calculated from (b) by increasing ADP concentrations as indicated in the Figure. There was a concentration-dependent relief of the ATP inhibition with ADP in WT and Kir6.2-K338E–SUR2A WT; however, this did not occur with Kir6.2 WT–SUR2A-E1318R. The relief of inhibition was restored when the complimentary charge swap mutant, Kir6.2-K3338E, was co-expressed with SUR2A-E1318R. (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant; repeated measures ANOVA with Bonferroni’s post-hoc test, n = 6 patches for each subunit combination).

current was obtained from KATP subunit combinations. This current was inhibited to 10 % of the peak current by titration with ATP (IC90 ). The fraction of ATP-inhibited current that could be relieved by incubation with different concentrations of ADP was then measured. Disruption of the Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge by the single charge reversal K338E in the Kir6.2 subunit had no effect on ADP sensitivity (Figure 8d). In contrast, single charge reversal in SUR2A-E1318R resulted in almost complete abolition of ADP sensitivity (Figure 8d). Re-establishment of a salt bridge between these residues in the double charge swap Kir6.2-K338E– SUR2A-E1318R combination restored ADP sensitivity to WT levels (Figure 8d). In keeping with the reduction in ATP sensitivity seen with the SUR2A-E1318R mutation alone, it seems likely that loss of ADP sensitivity was due to a change in the conformation of SUR2A. A salt bridge with function similar to that of Kir6.2-Lys338 –SUR2A-Glu1318 is present in Kir6.1–SUR2A channels

To confirm the importance of the Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge in KATP channel function, Kir6.1–SUR2A subunit combinations were expressed either before or following mutation of the equivalent charged residue in the Kir6.1 C-terminus, Arg347 , and sensitivity to pinacidil and glibenclamide was measured. Similar to Kir6.2-Lys338 , single charge reversal, Kir6.1-

R347E, expressed with WT SUR2A, resulted in channels with decreased EC50 for pinacidil (0.71 + − 1.2 μM compared with WT, 43.9 + − 1.3 μM, Figures 9a and 9b). Reinstatement of the salt bridge by charge reversals in both subunits (Kir6.1R347E–SUR2A-E1318R) moved the EC50 back towards WT (23.5 + − 1.3 μM, Figure 9b). Similarly, when glibenclamide sensitivity was considered, like Kir6.2-Lys338 , single charge reversal of Kir6.1-R347E resulted in an increase in IC50 (241.6 + − 1.1 nM), which was − 1.1 nM compared with WT, 6.2 + restored towards that of WT channels when single charge reversals in both subunits of residues involved in this salt bridge (Kir6.1-R347E–SUR2A-E1318R) were made (13.8 + − 1.1 nM) (Figure 9c). The close similarity of these results to those obtained after mutagenesis of the Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge and the conservation of charges at equivalent positions in all Kir6 and SUR isoforms confirmed the functional importance of an intersubunit electrostatic interaction at this position in KATP channel structure. DISCUSSION

We have characterized three novel electrostatic interactions, including a salt bridge, in KATP channels between the cytoplasmic C-terminal domain of Kir6.2 and NBD2 in the proximal Cterminal domain of SUR2A subunits. Disruption of native  c The Authors Journal compilation  c 2014 Biochemical Society

352

Figure 9

D. Lodwick and others

Kir6.1-R347E–SUR2A-E1318R charge swap also restores WT channel pharmacology

(a) Example recording of WT Kir6.1–SUR2A channel activation by 1, 10 and 100 μM pinacidil and inhibition with 10 μM glibenclamide. (b) Mean concentration–response data to pinacidil stimulation (normalized to cell capacitance) with Kir6.1 and SUR2A subunits as identified in the Figure. Kir6.1-R347E and SUR2A WT formed channels which had a marked increase in sensitivity + to pinacidil which was reversed by co-expression with the putative charge-swap partner SUR2A-E1318R (EC50 = 0.7 + − 0.1 μM compared with 44.9 − 8.1 μM for WT channels, P < 0.001, and 23.5 + − 13.4 μM with Kir6.1-R347E and SUR2A-E1318R, P > 0.05, ANOVA with Bonferroni’s post-hoc test, n  6 cells for each data point). (c) Mean concentration–inhibition data showing glibenclamide inhibition of pinacidil current activated using an approximate EC50 concentration. Kir6.1-R347E co-expression with SUR2A WT showed a leftward shift in concentration–inhibition + curve with an IC50 changing from 5.7 + − 0.1 nM to 480 − 27 nM (P < 0.0001). The IC50 was restored to WT by co-expression of the putative partner charge-swap residue in SUR2A (E1318R) 3.8 nM, P > 0.05, ANOVA with Bonferroni’s post-hoc test, n  5 cells for each data point). (IC50 = 9.3 + −

allosteric regulation in response to the channel activator pinacidil and the antagonist glibenclamide, and the near restoration of channel allostery, on breakage and repair of the salt bridge respectively, highlights the functional importance of this region of interaction between heterologous subunits. Interaction between TMD0 and the M2 TMD of Kir6 subunits has previously been deemed important for the assembly of the channel [29]. Moreover, isoform-specific regulation by nucleotides of channel opening has also been linked to the L0 linking sequence between TMD0 and TMD1 in SUR subunits [29–31]. The present data imply a further area of contact between heterologous subunits that is important in defining the functional and allosteric properties of the channel. Disruption of the Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge by single charge reversal in either subunit resulted in altered allostery from SUR2A to Kir6.2 in response to channel opener pinacidil and antagonist, glibenclamide, revealing an important functional interaction between these heterologous subunits. These changes occurred with only small concomitant changes in IC50 for ATP, indicating a direct disturbance of intersubunit information transfer, rather than an indirect effect resulting from altered affinity or efficacy of ATP binding to the Kir6.2 subunit. Reinstatement of the salt bridge by charge reversal in both Kir6.2 and SUR2A restored allostery from pinacidil and glibenclamide binding close to WT, indicating an important stabilizing contribution of this electrostatic contact between heterologous subunits. In contrast with pinacidil, Mg-ADP agonism was unaltered by breaking of the Kir6.2-Lys338 –SUR2AGlu1318 salt bridge, but was almost abolished in the SUR2A E1318R mutant. The apparently paradoxical reductions in pinacidil and ADP sensitivity in the E1318R substitution can be explained by the likely effect of this mutation on NBD2. Binding of KCOs to SUR has previously been shown to be dependent on ATP hydrolysis and mutations in the Walker-A and linker regions (including G1446R and K1352R) weaken or abolish KCO binding [32]. The replacement of Glu1318 with arginine would be expected to have a significant electrostatic effect and displacement of the Glu1318 loop could be transmitted to the ATP/ADP-binding site through Tyr1317 , which stacks on top of the adenine moiety (Figure 5c), and most probably also via the β-sheet residues, which connect the loop to the Walker-A motif.  c The Authors Journal compilation  c 2014 Biochemical Society

The role of SUR in nucleotide regulation of KATP is complex. In addition to the channel activation resulting from binding of nucleotides to the NBDs, SUR has an opposing role by constitutively enhancing the sensitivity of Kir6 to ATP; the ATP sensitivity of truncation mutants of Kir6.2 (which can express without SUR) is 10-fold higher when SUR is present than when it is absent [33]. This suggests that the presence of SUR affects either ATP binding to Kir6.2 or the ease with which this is translated into channel closure. Thus, SUR has both a structural and a regulatory role in channel regulation by ATP. Residues in SUR1 L0 and the Kir6.2 N-terminal cytoplasmic domain have been suggested to contribute to the structural role [34]. The Kir6.2Lys338 –SUR2A-Glu1318 salt bridge, showing proximity of SUR NBD2 and the C-terminal part of the Kir6.2 ATP-binding site, and the reduction in ATP sensitivity in the SUR2A-E1318R mutant, to a value intermediate to that of WT and a SUR-less Kir6.2 truncation mutant, suggests that this second region of SUR could also contribute to its structural influence on nucleotide regulation. The similarities in pharmacological response to salt-bridge disruption between Kir6.2 and Kir6.1 suggest a common interface, despite the regulatory differences between these subunits; Kir6.1containing channels differ from those with Kir6.2 in that they are relatively insensitive to ATP and they fail to open spontaneously in its absence. Interestingly, this apparent insensitivity to ATP results from an enhanced response to stimulatory ATP binding to SUR, rather than reduced sensitivity to inhibitory binding to Kir6.1 itself [35]. On the basis of our previous identification of a Kir6 interaction motif within SUR2A [19], Dupuis et al. [36] identified three SUR2A residues, Glu1305 , Ile1310 and Leu1313 (cf. Figures 6b and 6c) which, when changed together to those of non-interacting MRP1, were sufficient to attenuate significantly the activation of KATP channel activity by Mg-ADP and potassium channel openers from SUR2A. These three residues map to the strand β1, which is structurally connected to the three charged residues identified herein, suggesting that the β1–β2 strands form the interface transmitting activation from SUR2A to Kir6.2. It is noteworthy that glibenclamide inhibition was unaffected by mutations of Glu1305 , Ile1310 and Leu1313 [36], suggesting that, although there is some overlap, the interfaces transmitting information from agonist and antagonist are not identical.

Allosteric regulation of KATP channels

It is of interest that deletion of Phe1388 in SUR1, a mutation identified in congenital hyperinsulinaemia, results in defective stimulatory responses to Mg-ADP and diazoxide and disrupted trafficking of channels to the cell surface [37]. Single amino acid substitutions in this region showed that hydrophobicity was important for correct trafficking but that the detailed architecture of the residue in this position was more important for determining sensitivity to Mg-ADP and KATP channel drugs. Indeed, the conservative substitution of leucine at this position (Leu1351 in SUR2A and SUR2B) was suggested to explain some of the pharmacological differences between SUR isoforms [37]. In SUR2A, due to a β1-bulge in strand β1, Leu1351 , Ile1310 and Leu1313 form a tightly packed hydrophobic core (Figure 6c), thus directly connecting the β1–β2 interface with the α-helical region which comprises part of the Walker-A nucleotide-binding motif. This arrangement could function as the mechanism for the transfer of allosteric information from nucleotide- and drug-binding sites on SUR2A to the ATP-binding, cytoplasmic domain in Kir6.2. It has been suggested that sulfonylureas, such as ATP, stabilize the closed state of KATP channels [38,39]. Truncation of the N-terminus of Kir6.2 blocked coupling between sulfonylurea binding to SUR and stabilization of the closed state of the channel. Conversely, potassium channel openers may stabilize the open state (but with no requirement for the Kir6.2 N-terminus) [39]. Disruption of the Kir6.2-Lys338 –SUR2A-Glu1318 salt bridge caused increased pinacidil sensitivity and reduced glibenclamide sensitivity. This could indicate a stabilization of the open state of mutant channels (similar to that seen in the Kir6.2-L164A mutant [39]), but this cannot be the case as the ATP sensitivity of the K338E mutant was unchanged. This suggests that the baseline properties of the channel were maintained but that transduction of the conformational changes induced by drug binding was altered. The combination of enhanced pinacidil and reduced glibenclamide sensitivity could arise from increased nucleotide binding or hydrolysis; mutations that compromise either reduce pinacidil sensitivity [32] and it has been suggested that sulfonylureas interfere with the stimulatory action of nucleotides [38]. However, neither of these possibilities is consistent with the unchanged ADP and ATP sensitivities of the K338E mutant, thus underlining the role of this salt bridge interface in the transduction of regulatory information initiated by conformational changes in response to drug binding to SUR. In summary, an interface between heterologous KATP channel subunits was identified containing an electrostatic interaction between the Kir6.2-Lys338 and SUR2A-Glu1318 residues, which is important for determining channel sensitivity to various allosteric regulators.

AUTHOR CONTRIBUTION Robert Norman, David Lodwick and Richard Rainbow designed the experiments, which were performed by Richard Rainbow and Hussein Rubaiy (electrophysiology), Hussein Rubaiy, Mohammed Al Johi and Robert Norman (biochemistry), Geerten Vuister (structural biology) and David Lodwick, Mohammed Al Johi and Hussein Rubaiy (molecular biology). Robert Norman, David Lodwick, Richard Rainbow and Geerten Vuister wrote the paper.

ACKNOWLEDGEMENTS We thank Professor Andrew Tinker and Professor Asipu Sivaprasadarao for Kir6.2–Kir2.1 chimaeras, Professor Susumu Seino for Kir6.1 and SUR2A clones and Professor Yoshihisa Kurachi for a Kir6.2 clone. We thank Sonja Khemiri, Barbara Horley and Gwyneth Williams for expert technical assistance.

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FUNDING This work was supported by the British Heart Foundation [project grant number 98011 (to R.I.N. and D.L.)] and studentships to M.A.J. (Royal Saudi Embassy) and H.N.R. (TH Wathes).

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 c The Authors Journal compilation  c 2014 Biochemical Society

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