Báo cáo Y học: The cytoplasmic C-terminus of the sulfonylurea receptor is important for KATP channel function but is not key for complex assembly or trafficking pdf

Eur J Biochem 269, 5303–5313 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03249.x The cytoplasmic C-terminus of the sulfonylurea receptor is important for KATP channel function but is not key for complex assembly or trafficking Jonathan P Giblin*, Kathryn Quinn* and Andrew Tinker Centre for Clinical Pharmacology, Department of Medicine, University College London, The Rayne Institute, UK ATP-sensitive K+ channels are an octameric assembly of two proteins, a sulfonylurea receptor (SUR1) and an ion conducting subunit (Kir 6.0) We have examined the role of the C-terminus of SUR1 by expressing a series of truncation mutants together with Kir6.2 stably in HEK293 cells Biochemical analyses using coimmunoprecipitation indicate that SUR1 deletion mutants and Kir6.2 assemble and that a SUR1 deletion mutant binds glibenclamide with high affinity Electrophysiological recordings indicate that ATP sensitivity is normal but the response of the mutant channel complexes to tolbutamide, MgADP and diazoxide is dis- turbed Quantitative immunofluorescence and cell surface biotinylation supports the idea that there is little disturbance in the efficiency of trafficking Our data show that deletions of the C-terminal most cytoplasmic domain of SUR1, can result in functional channels at the plasma membrane in mammalian cells that have an abnormal response to physiological and pharmacological agents ATP-sensitive potassium channels (KATP) are present in the plasma membrane of a number of tissues and are also present in endomembranes such as mitochondria They have been proposed to be involved in a number of physiological and pathophysiological processes and form a link between cellular metabolism and membrane excitability For example, in the pancreas, KATP regulates insulin release and in vascular smooth muscle, it is regulated by vasodilators and influences blood flow in certain vascular beds KATP is an octameric protein complex composed of two subunit types namely a pore forming subunit (Kir6.1, Kir6.2), a member of the inwardly rectifying family of K+ channel, and the sulfonylurea receptor subunit, a member of the ATP binding cassette family of proteins (SUR1, SUR2A, SUR2B) The assembly of a particular pore forming subunit with a particular SUR generates currents with a characteristic single-channel conductance, nucleotide regulation and pharmacology [1–4] Kir subunits have a cytoplasmic N and C terminus with two transmembrane domains and a pore forming H5 loop [5,6] SUR has multiple transmembrane domains with two large intracytoplasmic loops, the first and second nucleotide binding domains (NBD1 and NBD2), which contain consensus sequences for the hydrolysis of nucleotides (Walker A and B motifs) [7,8] The trafficking of the KATP channel complex has been the subject of some investigation It was initially observed that coexpression of the two proteins was necessary to generate significant plasmalemmal currents [9] A series of studies have supported the idea that the Kir6.2 and SUR1 subunits have small peptide motifs (RKR) that either prevent the export of the protein from the ER and/ or retrieve it from the Golgi [10] The simultaneous masking of these two signals by interaction of the two proteins allows the channel complex to proceed through the biosynthetic pathway to the membrane It has also been suggested that the most distal part of the C-terminus of SUR1 contains an anterograde signal that allows export from the ER [11] Potentially, this has clinical ramifications as a number of mutations in persistent hyperinsulinaemic hypoglycaemia of infancy [12] cluster in this domain of the protein Persistent hyperinsulinaemic hypoglycaemia of infancy is an hereditary disease characterized by inappropriately high levels of insulin release and hypoglycaemia in children at birth It is hypothesized that deletion of the most distal part of the C-terminus (only seven amino acids) leads to the removal of a forward trafficking signal and retention in the ER A phenylalanine (1574) and a leucine (1566) were established as being particularly important However a splice variant of SUR1 has been described and cloned from a hypothalamic cDNA library that removes exon 33 and results in a frameshift and premature termination of the C-terminus preceding the Walker A and B motifs [13] Two other deletions were constructed at the beginning and end of the C-terminus in exon 33 All these were functional upon expression and the truncation of 253 amino acids did not affect the magnitude of macroscopic currents In a related vein, a recent report has shown a SUR1-MRP1 C-terminal chimaera is able to traffic to the plasma membrane when coexpressed with Kir6.2 [14] Correspondence to A Tinker, Room F2, 4th Floor, Centre for Clinical Pharmacology, Department of Medicine, University College London, The Rayne Institute, University Street, London WC1E 6JJ, UK, Fax: + 44 20 76912838, Tel.: + 44 20 76796192, E-mail: a.tinker@ucl.ac.uk *Note: These authors contributed equally to this work (Received July 2002, revised 19 August 2002, accepted 11 September 2002) Keywords: K channel; inward rectifier; ATP-sensitive; trafficking and assembly; sulfonylurea receptor Ó FEBS 2002 5304 J P Giblin et al (Eur J Biochem 269) There are several potential explanations for these different results Firstly, there are differences in the expression system used (Cos cells in [11] vs Xenopus laevis oocytes in [13] and [14]) and secondly the details of the deletions vary To try to resolve these differences we have constructed a series of deletions in the second nucleotide binding domain, expressed and studied their biochemical and functional behaviour in a human kidney cell line (HEK293 cells) MATERIALS AND METHODS Molecular biology Standard subcloning techniques were used throughout A SUR1 mutant with a myc epitope was used as previously described [15] In our initial studies we constructed a 146 amino acid deletion of SUR1 with the myc epitope The pcDNA3 vector was digested with NotI/ApaI Sense and antisense oligonucleotides corresponding to an artificial gene fragment encoding the myc epitope and stop codon together with compatible overhangs were annealed and ligated into pcDNA3 A NotI fragment of SUR1 (from SUR1 in pcDNA3) was then subcloned into this construct Subsequently a range of SUR1 deletions, tagged with the 10 amino acids constituting the myc epitope, were constructed from SUR1 in pBluescript (SK–) A two stage PCR strategy was used to generate a deletion cassette with a 5¢-SfiI site in the clone and the myc epitope followed by a stop codon and an AvrII site The native fragment was replaced by this cassette and resulting product subcloned into the XhoI/XbaI sites of pcDNA3 using a SalI/SpeI digest Kir6.2 and Kir6.2mycHis6 was expressed in pcDNA3.1/Zeo (Invitrogen) Kir6.2mycHis6 was generated from a previous study [15] The sequence of all mutants was confirmed by DNA sequencing using the dRhodamine Terminator cycle sequencing kit (Applied Biosciences) and an automatic sequencer (ABI 377, Perkin-Elmer) Cell culture and transfection HEK293 cells were cultured, transfected and stable cell lines expressing Kir6.2, Kir6.2mycHis6 and SUR1-myc and deletion mutants generated as previously described [15] A number of new monoclonal lines have been generated in this study and they are detailed as appropriate in the text For selection with G418 we used 727 lgỈmL)1, for Zeocin 364 lgỈmL)1 and in combination 727 lgỈmL)1 G418 and 364 lgỈmL)1 Zeocin Antiserum production in rabbits A peptide corresponding to amino acid residues 942–955 of hamster SUR1 (ETVMERKASEPSQGC, final cysteine added for coupling purposes) was synthesized and linked to keyhole limpet haemocyanin before injection into rabbits using standard protocols (Regal Group Ltd, Great Bookham, Surrey, UK) Bleeds were assayed for activity using an antibody capture assay [16] and bleeds showing reactivity were affinity purified Kir6.2 antisera to the C-terminus of the channel and myc hybridoma cells were used as previously described [15,17] Gel electrophoresis, radioligand binding, immunoprecipitation and immunofluorescence SDS/PAGE, Western blotting, radioligand binding, immunoprecipitation and immunofluorescence staining were carried out as previously described [15,17] with some modifications for colocalization experiments In colocalization experiments slides were incubated with the first primary antibody [a : 500 dilution of anti-(Kir6.2 C-terminus) Ig] and the appropriate secondary antibody (a : 300 dilution of a rhodamine-linked secondary goat anti-rabbit Ig) for h each as previously described [17] After washing, the second primary antibody was applied overnight (a : 500 dilution of anti-myc Ig) and the appropriate secondary antibody (a : 300 dilution of a fluorescein-linked secondary goat anti-mouse Ig) was then applied the following day for h The antibody reactive to the Kir6.2 C-terminus was raised to the peptide sequence DALTLASSGPLRKRSC and has been characterized previously [17] The anti-myc Ig used was purified on Protein A Sepharose CL-4B (Amersham-Pharmacia biotech) from mouse 9E10 hybridoma cell line culture supernatant under high salt conditions using a standard method (p311; [16]) All fluorophore conjugated secondary antibodies were purchased from Molecular Probes Inc Gel densitometry was performed using SCION IMAGE as detailed in the help section in the program For the imaging work, slides were viewed and analysed using a computer based image analysis system (OPENLAB 3.1, Improvision) coupled to a Zeiss Axiovert 100M microscope set up for epifluorescence equipped with an appropriate filter set that allowed imaging of both fluorescein and rhodamine fluorophores (XF66-1 multiband filter, Omega optical) Images used for quantitative analysis and colocalization experiments represented a focal plane taken through the middle of the cell Scattered and out-of-focus fluorescent signal was removed from the image by deconvolution The image was deconvolved from a z-stack of 31 images with 0.2 lm spacing using the OPENLAB software (volume deconvolution module) The quantitative assay was performed on the deconvolved images using the OPENLAB advanced measurements module The theory behind the quantitative assay is given in Fig 6A In colocalization experiments, deconvolved images of the cell at each excitatory wavelength were obtained and merged using the OPENLAB software Procedure for surface biotinylation Stable cell lines were cultured in 100 mm2 tissue culture dishes as previously described [15] until 75–95% confluent Each dish was washed three times with ice-cold phosphate buffered saline (NaCl/Pi – 10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4, prepared from tablets supplied from Sigma, Poole, UK) before incubation with mL 0.5 mgỈmL)1 EZ-Link Sulfo-NHS-LCBiotin (Pierce, Rockford, Illinois, USA) in NaCl/Pi at °C for 30 Dishes were washed twice with ice-cold NaCl/Pi before incubation with NaCl/Pi + 100 mM glycine at °C for 20 to quench any remaining unreacted biotin reagent Cells were then lysed by scraping into 250 lL 1% (w/v) SDS in Tris buffered saline (Tris/ NaCl – 50 mM TrisHCl, 150 mM NaCl, mM KCl, Ó FEBS 2002 Role of the cytoplasmic C-terminus of SUR1 (Eur J Biochem 269) 5305 pH 7.4) containing mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Roche Complete EDTA-free, Roche Diagnostics Limited, Lewes, Sussex, UK) The lysates were subsequently incubated at 65 °C for 10 before dilution with mL 1% (v/v) Triton in Tris/NaCl containing mM phenylmethanesulfonyl fluoride and the protease inhibitor cocktail Lysates were then incubated on ice for h before being briefly sonicated (2 s using an MSE Soniprep 150 probe sonicator at half power) Sonicated lysates were centrifuged at 20 000 g for 30 to pellet any insoluble material and biotinylated proteins were isolated from the supernatant as described below A 50-lL sample was removed at this point and used for subsequent SDS/PAGE and Western blotting analysis Isolation of surface biotinylated proteins The supernatant obtained from the surface biotinylation procedure was incubated with 150 lL of a pre-equilibrated : slurry of Ultra-link Immobilized NeutrAvidin biotin binding protein (Pierce) overnight at °C with gentle rotation The slurry was pre-equilibrated with binding buffer [1% (v/v) Triton, 0.20% (w/v) SDS in Tris/NaCl] After the incubation period, the binding resin was pelleted by centrifugation (20 000 g for at °C) followed by five washes with mL binding buffer Bound protein was recovered by incubation with 80 lL · Laemmli gel loading buffer (350 mM TrisHCl pH 6.8, 10.28 (w/v) SDS, 36% (v/v) glycerol, 0.012% (w/v) bromophenol blue, 200 mM dithiothreitol) at 100 °C for Eluted proteins were subsequently analysed by SDS/PAGE followed by Western blotting Electrophysiology Whole-cell and inside-out patch clamp recordings were performed using an Axopatch 200B amplifier (Axon Instruments) and were digitized using a Digidata 1200 interface before capture to a computer hard disk Whole-cell current signals were filtered at kHz and sampled at kHz, and analysed using PCLAMP6 software (Axon Instruments) Unitary single-channel currents were filtered at kHz and sampled at kHz, and analysed to determine either mean NPo (Number of channels · Open probability) during a 30 second sweep (using pClamp6), or mean current (using Satori, Intracel Ltd) Patch pipettes were pulled using a PP830 pipette puller, and fire-polished using a MF-830 microforge (both Narishige) Pipettes had resistances of 1.5–3 MW for whole-cell recording and 6–9 MW for single-channel recordings The capacitance of pipettes was reduced by coating pipettes with a parafilm/mineral oil suspension and compensated for by using the amplifier Series resistance during whole cell recording was compensated to at least 70% using amplifier circuitry The pipette/ bath solution contained in mM; 107 KCl, 1.2 MgCl2, CaCl2, 10 EGTA, Hepes (with 33 mM KOH to pH 7.2) and the bath/pipette solution; 140 KCl, 2.6 CaCl2, 1.2 MgCl2, Hepes (pH 7.4) for whole cell/single-channel work Whole-cell pipette solutions were supplemented with ATP and ADP, and pH was re-adjusted to 7.2 (see figure legends for nucleotide concentrations used in each stable line) Data analysis Radioligand binding experiments were fitted by the binding isotherm y ẳ Bmaxặx/(x + Kd) where y is the bound specific radioligand (pmolỈmg protein)1) and x is the radioligand concentration For dose–response curves with inhibition of current by tolbutamide the data from individual experiments were expressed and fitted to: % inhibition of maximal current ¼ a + (b ) a)/(1 + (x/Ki)h) where b was fixed at zero and a represents the limiting inhibition (expressed as percentage inhibition of maximal current), h the hill coefficient and Ki the EC50 for inhibition Mean Ki was calculated by fitting individual experiments with the curves and calculating mean parameters as indicated Statistical analysis was carried out using one-way ANOVA with an appropriate posthoc test or Students t-test as appropriate (ORIGIN v6.0 and PRISM v3.0) Statistical significance is as indicated in the legend and text Data are presented as mean ± SEM RESULTS To facilitate biochemical studies, we generated a polyclonal rabbit antisera raised to a short peptide sequence in the first nucleotide binding domain of SUR1 (see Materials and methods) Figure 1A and B show the characterization of the specificity of the antisera for immunoblotting and immunofluorescence, respectively It is apparent that the antisera recognizes bands of the correct molecular mass in a SUR1 + Kir6.2 stable line (a band at  150 kDa and another at  170 kDa) but not in stable lines expressing SUR2A + Kir6.2, SUR2B + Kir6.1 and wildtype nontransfected HEK293 cells (Fig 1A) The antisera also recognizes a number of other proteins of lower molecular mass endogenous to HEK293 cells however, this does not influence the nature of our conclusions below When assayed using immunofluorescence, the antisera reacted with a stable line expressing SUR1 + Kir6.2 but not one expressing SUR2B + Kir6.1 (Fig 1B) The signal was competed with by the immunogenic peptide (Fig 1B) and incubation with the secondary alone led to no signal (not shown) Generation of SUR1 deletions and stable cell lines Using standard molecular cloning methods (see Materials and methods) we generated a series of SUR1 deletions tagged with the 10 amino acid myc epitope (Fig 2A) In our initial studies we first examined a 146 amino acid deletion (SUR1del146myc) and subsequently engineered a series of these We generated a polyclonal stable cell line in HEK293 cells stably expressing SUR1del146myc by selection with G418 and stable monoclonal cell lines expressing Kir6.2 + SUR1myc (line A), Kir6.2 + SUR1del101myc (line B), Kir6.2 + SUR1del145myc (line C1) and Kir6.2mycHis6 + SUR1del146myc (line C2), Kir6.2 + SUR1del196myc (line D) and Kir6.2 + SUR1del249myc (line E) with G418 and Zeocin as previously described [15,18] Lines were screened using biochemical and subsequently electrophysiological methods Line C1 was labile with multiple passages often losing current Line C2 was more stable and could be passaged for long periods without 5306 J P Giblin et al (Eur J Biochem 269) Ó FEBS 2002 Fig Characterization of SUR1 antibody by Western blotting and immunofluorescence The Western blot in (A), probed with a : 2000 dilution of anti-SUR1 Ig, is derived from an 8% polyacrylamide gel Lanes were loaded with lg of cellular homogenates of the stable lines indicated WT denotes untransfected HEK293 cells The positions of molecular mass markers (in kDa) are indicated to the left of the blot and the position of SUR1 is indicated by the arrow Note that SUR1 migrates as a doublet with bands of approximately 150 and 170 kDa in size The blot represents an exposure to film of 30 s The images in (B) show images of cells from the stable lines indicated stained with a : 500 dilution of anti-SUR1 Ig Reactivity is observed against cells expressing SUR1 but not against cells expressing SUR2B SUR1 reactivity is abolished by preincubation of the antibody with mgỈmL)1 antigenic peptide for h The rhodamine-linked secondary antibody was used at a : 300 dilution All images were captured at the same exposure (1 s) and magnification The scale bar represents lm loss of current As a result most electrophysiological studies were performed on this line Kir6.2 interacts with SUR1 and the SUR1 deletions We used a coimmunoprecipitation strategy to examine the interaction of Kir6.2 with SUR1-myc and SUR1-myc deletions Immunoprecipitation of SUR1-myc and deletion constructs was performed using a mouse monoclonal antibody to myc to avoid problems with discrimination between the rabbit immunoglobulin heavy chain and Kir6.2 that might occur if the precipitating antibody were rabbit in origin Immunoprecipitation of SUR1 was confirmed by probing with the rabbit polyclonal antisera to SUR1 Figure 2B shows the coimmunoprecipitation of Kir6.2 with SUR1-myc and each of the deletions The upper blots show the level of protein expression in the selected lines prior to immunoprecipitation Kir6.2 is detected using a rabbit polyclonal antisera raised to the C-terminus of the protein (see Materials and methods and [17]) Controls for the immunoprecipitation protocol have previously been shown [15] We further showed the close association and localization of Kir6.2 and SUR1-myc by costaining cells with the myc mouse monoclonal antibody and the rabbit anti-(Kir6.2 C-terminus) Ig In addition, we also examined the colocalization of Kir6.2 with the most profound deletion SUR1del249myc In Fig 2C it is apparent that the two signals colocalize in both line A and line E Thus the ability of Kir6.2 to assemble with SUR1 is not grossly affected by deletion of the second nucleotide binding domain Functional properties of the deletion mutants coexpressed with Kir6.2 Electrophysiological techniques revealed that all the deletion mutants were able to express significant currents at the plasma membrane of HEK293 cells In Fig 3A and B representative traces are shown from whole cell patch clamp recordings from line A (Ai and Aii) as compared to the most profound deletion in line E (Bi and Bii) After rupture of the membrane to gain access to the intracellular contents, whole-cell currents increased to a steady-state value The magnitude of this was dependent on the pipette ATP concentration Currents in line A were substantially inhibited by 100 lM tolbutamide, whereas those in line E were only partially inhibited (see below) Both currents were completely inhibited at hyperpolarized potentials by 10 mM Ba2+ (Fig 3Aii and Bii) All the SUR1 deletions in the monoclonal stable lines gave rise to currents significantly in excess of those from wildtype cells [15] However the magnitude of these varied with clonal isolates and some lines possessed smaller currents than others With 1.2 mM ATP in the patch pipette, the current density at )50 mV was: in line A ¼ 586 ± 117 pA/pF (n ¼ 14); in line B ¼ 9.5 ± 2.1 pA/pF (n ¼ 11); in line D ¼ 51.2 ± 14.1 pA/pF (n ¼ 7) and in line E ¼ 492 ± 136 pA/pF (n ¼ 11) With 0.6 mM ATP in the patch pipette, the current density at )50 mV in line C2 was 313 ± 44 (n ¼ 8) pA/pF This electrophysiological behaviour was reflected in variations in the expression of the relevant components as determined by immunoblotting Figure 3C shows representative recordings of single-channels in the inside-out configuration Ó FEBS 2002 Role of the cytoplasmic C-terminus of SUR1 (Eur J Biochem 269) 5307 Fig Assembly of SUR1 C-terminal deletion mutants with Kir6.2 The scheme in (A) shows the boundaries of the C-terminal deletion mutants of SUR1 The putative first nucleotide binding domain (NBD1) lies between amino acid residues 697–895 and the putative second nucleotide binding domain (NBD2) between residues 1359–1582, as indicated by the shading The Walker A and B consensus sequences in NBD2 are located between residues 1379 and 1385 and residues 1503–1507, respectively The upper panels of Western blots (B) show the expression of the SUR1 deletion mutants and Kir6.2 in each of the monoclonal lines indicated Two species of SUR were observed corresponding to putatively immature and maturely glycosylated forms Two bands were also sometimes observed for Kir6.2, the smaller band possibly representing a proteolytic fragment or a partially processed form Cell line homogenate (8 lg) was loaded into each lane WT represents a lane loaded with lg of nontransfected cell homogenate The lower set of blots (B) show the results of coimmunoprecipitation experiments performed on 0.8 mg of solubilized cell line homogenate The myc monoclonal antibody was used to immunoprecipitate myc-tagged SUR mutants Immunoprecipitation of SUR1 mutants with concomitant immunoprecipitation of Kir6.2 was observed for all lines tested, indicating that the C-terminus of SUR1 is not required for biochemical interaction with Kir6.2 Lanes were loaded with 50% of the total eluate and blots were probed with the anti-SUR1 and anti-Kir6.2 Ig All blots shown represent a 30 s exposure to photographic film Blots were probed with the antibodies as indicated The SUR1 and Kir6.2 antibodies were both used at a : 2000 dilution SUR1 and Kir6.2 were resolved on and 12% polyacrylamide gels, respectively The images in (C) show colocalization of Kir6.2 and SUR1myc (line A) and Kir6.2 and SUR1del249myc (line E) SUR1 was detected using the myc mouse monoclonal antibody purified with Protein A Sepharose (1 : 500 dilution – see Materials and methods) in conjunction with a fluorescein anti-mouse secondary Ig Kir6.2 was detected using the polyclonal rabbit anti-(Kir6.2 C-terminus) Ig (1 : 500 dilution) in conjunction with a rhodamine-linked anti-rabbit secondary Ig Cells were imaged at each excitatory wavelength and the resulting images overlaid using the imaging software The green scale bar represents lm The images shown represent deconvolved images (see Materials and methods) 5308 J P Giblin et al (Eur J Biochem 269) Ó FEBS 2002 Fig Examples of Kir6.2/SUR1 and NBD2 deletion currents in stably transfected HEK293 cells (Ai) Voltage-clamp recording of line A currents, and (Bi) voltage-clamp recording of line E currents, evoked during 250 ms voltage steps between )100 mV and +100 mV in 10 mV increments from a holding potential of mV Currents were blocked by 100 lM tolbutamide, and the block was reversed on subsequent washout (Aii) Voltage clamp recording of line A current, and (Bii) voltage-clamp recording of line E current, evoked during a 2-s ramp between )100 mV and +100 mV, showing block by 10 mM BaCl2 (dashed line), which was reversible on washout (C) Inside-out patch recordings of single-channel currents through stably expressed channels at a holding potential of )60 mV, with inhibition of channel activity by 100 lM ATP shown in both line A (Ci) and line E (Cii) All currents were recorded in symmetrical 140 mM K+, and whole cell recordings carried out with 1.2 mM ATP in the patch pipette Fig Effects of deletions in the NBD2 domain of SUR1 on channel sensitivity to glibenclamide, diazoxide and tolbutamide (A) Representative radioligand binding curve from a single experiment Points are specific binding activity (bars indicate SD of triplicate results) In this particular example, the best fit binding isotherm has a Kd ẳ 1.47 nM and Bmax ẳ 30.1 pmolặmg protein)1 (B) Effects of 100 lM tolbutamide on currents in lines A, B, C2, D and E, expressed as percentage inhibition of maximal current, with 1.2 mM ATP in the patch pipette for lines A and E, 600 lM ATP for line C2 and 100 lM ATP for lines B and D (C) Concentration–response curves for the effect of tolbutamide (1–500 lM) on A (d), E (j) and C2 (n) currents, expressed as percentage inhibition of maximal current Figures for Ki, h and a in text Pipette ATP concentrations as in (B) All currents recorded at )60 mV in symmetrical 140 mM K+ (D) Effect of 10 perfusion with 300 lM diazoxide on currents in lines A, B, C2, D and E Currents expressed as I/Ic, with mM ATP in the patch pipette in lines A, E and D and 1.2 mM ATP in the pipette in lines B and C2 Oneway ANOVA using Dunnett’s multiple comparison test was used to test for a significant difference in the effects on lines B-E, compared with A, where * shows P < 0.05 and ** shows P < 0.001 Numbers in brackets denote number of samples Ó FEBS 2002 Role of the cytoplasmic C-terminus of SUR1 (Eur J Biochem 269) 5309 It shows the inhibition of channels in line A and E by ATP The deletion of the C-terminus of SUR1 does not substantially affect the response to this nucleotide (Fig 3Ci and ii) and quantitation is shown in Fig 5B However other electrophysiological and pharmacological parameters were disturbed The interaction of the channel complex with the sulfonylurea class of drug was examined next We first performed radioligand binding with tritiated glibenclamide to the polyclonal SUR1del146myc cell line The Kd for drug binding is not significantly changed: for SUR1del146myc Kd ¼ 2.02 ± 0.35 nM (n ¼ 6) (a representative example is shown in Fig 4A) and for SUR1-myc as previously published [15] Kd ¼ 1.25 ± 0.17 nM (n ¼ 4) (not significant, P ¼ 0.12 unpaired t-test after logarithmic transformation) Next, we examined the electrophysiological effect of tolbutamide using the whole cell configuration of the patch clamp (Fig 4B.C) Concentrations of tolbutamide (100 lM) known to act selectively by binding to SUR1 and not to have significant pore blocking effects were used The lines containing the SUR1 deletions had currents that were reduced but not to the same extent compared to control Figure 4B summarizes the percentage inhibition of current in these lines Thus progressive deletion of the C-terminus of SUR1 affects the efficacy of drug response To further characterize this, dose–response curves were constructed for two of these and it was found that the Ki was not significantly affected once the differences in limiting value were taken into account The Ki for tolbutamide inhibition of the current in line A was 8.1 ± 3.3 lM and h ¼ 1.45 ± 0.1 and a ¼ 87.3 ± 3.9 (n ¼ 10), compared with 18.7 ± 7.5 lM, h ¼ 1.63 ± 0.3 and a ¼ 51.3 ± 4.0 for line C2 (n ¼ 6) and 6.2 ± 2.8 lM, h ¼ 1.43 ± 0.33 and a ¼ 33.0 ± 4.2 for line E (n ¼ 8) (for Ki P ¼ 0.09 after log transformation for all comparisons, one way ANOVA with Bonferronni correction) Figure 4C shows the mean pooled data at each point fitted with the best-fit curve (to the means) using nonlinear regression The resultant parameters are very similar but not identical to those determined after analysis of each individual experiment The latter however, allows statistical analysis of the relevant data The response to bath applied diazoxide was determined in the whole-cell configuration The application of 300 lM diazoxide induced large whole-cell currents in line A but led to little increase in the other lines (Fig 4D) Recent studies have shown an interrelationship between diazoxide stimulation and MgADP stimulation [19,20] Thus we examined bath applied MgADP in inside-out patches on line A and line E In the former line there was pronounced stimulation whilst in the latter there was a much more modest stimulation (Fig 5A and B) We also performed analogous experiments with line C2 and observed very little stimulation with MgADP (not shown) In summary, the deletion of the C-terminus of SUR1 results in functional channels that cannot be stimulated by diazoxide and MgADP The affinity of sulfonylurea interaction is not affected, however, the net resultant inhibitory effect is decreased as the C-terminus is progressively deleted Membrane trafficking studied using immunofluorescence The presence of significant membrane currents is an indication that trafficking is not significantly impaired Fig Effects of deletions on channel sensitivity to ADP (A) examples of inside-out patch recordings of single-channel currents through stably expressed channels, with unitary single-channel A currents shown in (Ai) and unitary single-channel E currents shown in (Aii) Both records show the effects on channel activity of application of 100 lM (Mg)ATP and 500 lM (Mg)ADP (B) Comparison of normalized (I/Ic) single-channel currents between A (n ¼ 9) and E (n ¼ 5) in the presence of 100 lM ATP and 100 lM ATP with 500 lM ADP Student’s paired t-test used to test for a significant change in normalized NPo on addition of ADP, where * ¼ P < 0.05 All currents recorded at )60 mV in symmetrical 140 mM K+ However the possibility of differences in the relative level of expression of the different mutants may confound such a picture For example, it is possible for there to be significant membrane currents but for a significant fraction of protein to be retained intracellularly A number of elegant and imaginative methods have been developed to examine these problems [10,21]; however, they suffer from the complication that calibration for relative expression must be obtained independently We took a different approach The fraction of immunofluorescent signal at or close to the plasma membrane was calculated in a number of cells First a mask was defined round the edge of the cell and then a further mask was defined (performed by shrinking the original mask by 0.4 lm in each direction), which excludes the membrane and membrane associated area but includes the intracellular contents The total fluoresence in each situation is calculated from mean pixel intensity multiplied by the number of pixels The ÔmembraneÕ associated fluoresence is calculated by subtracting the two and the ÔmembraneÕ 5310 J P Giblin et al (Eur J Biochem 269) signal is normalized to the total fluorescence Potentially this controls internally within each cell for differences in expression The principle of the assay is shown in Fig 6A The sensitivity of such an assay was tested in detecting the membrane translocation of SUR1 and Kir6.2 when expressed independently and in the situation where they are coexpressed To facilitate these studies we generated stable lines expressing Kir6.2 with Zeocin selection and SUR1 with G418 (see Materials and methods) The fraction of immunofluorescence associated with the membrane was compared between SUR1 and SUR1 + Kir6.2 stable lines probed with the anti-SUR1 Ig and between Kir6.2 and Kir6.2 + SUR1 stable lines probed with the anti-(Kir6.2 C-terminus) Ig Representative images and the quantitative data are shown in Fig 6B–D In both cases there was a measurable and significant increase in membrane associated immunofluorescence in keeping with the visual appearance of the images (see Discussion) The fraction retained upon single expression of a subunit (SUR1 or Kir6.2) and the increase upon coexpression was approximately the same when detected by either antibody We then used this approach to examine the trafficking of Kir6.2 in lines A and line E Representative images and the Ó FEBS 2002 quantitative data are shown in Fig 7A and B We were unable to detect a significant difference in trafficking of Kir6.2 when coexpressed with full length SUR1-myc and the most profound deletion, SUR1del249myc In other words even with the most profound deletion there is no gross alteration in the efficiency of trafficking as assessed using immunofluorescence As our assay will not distinguish between protein located at the membrane or just below it, we used a further experimental approach to examine this question We labelled membrane proteins with biotin using a cell impermeable reagent (see Materials and methods) and purified the resulting products using an avidin derivative complexed to a solid support A sample of the cellular lysate prior to purification and samples after purification are then subjected to Western blotting with antibodies to SUR1 and Kir6.2 Figure 8A shows that only small amounts of SUR1 are labelled when expressed in a stable cell line alone However, coexpression of SUR1-myc with Kir6.2 in line A results in significant labelling Furthermore, coexpression of Kir6.2 with the most profound deletion, SUR1del249myc, in line E results in comparable signal We also noted that Kir6.2 was purified under such Fig Study of KATP channel subunit trafficking using immunofluorescence The scheme in (A) shows the principle of the quantitative trafficking assay The images in (B) show representative deconvolved images of cells expressing either Kir6.2 or Kir6.2 + SUR1 stained with a : 500 dilution of anti-(Kir6.2 C-terminus) Ig The images in (C) show representative deconvolved images of cells expressing either SUR1 or Kir6.2 + SUR1 stained with a : 500 dilution of anti-SUR1 Ig An anti-rabbit rhodamine-linked secondary Ig was used to detect bound primary antibody Note the increase in membrane associated staining when both subunits are coexpressed The graphs in (D) show the data from the quantitative trafficking assay It can be observed that coexpressing of channel subunits significantly changes the percentage of membrane associated fluorescence corresponding to both Kir6.2 and SUR1 The scale bar on the images represents lm Ó FEBS 2002 Role of the cytoplasmic C-terminus of SUR1 (Eur J Biochem 269) 5311 Fig Analysis of the effect of coexpression of SUR1del249myc on Kir6.2 trafficking The images in (A) show representative deconvolved images of cells coexpressing either Kir6.2 + SUR1myc (line A) or Kir6.2 + SUR1del249myc (line E) stained with a : 500 dilution of the anti-(Kir6.2 C-terminus) Ig Note the presence of membrane associated staining in both images The scale bar on the images represents lm The data obtained from the quantitative trafficking assay is shown in (B) There is no significant difference in the percentage of fluorescence associated with the membrane when Kir6.2 is coexpressed with either SUR1myc or SUR1del249myc conditions (Fig 8B) and due to the presence generally of a single distinct band was better suited to quantification Thus assuming a high efficiency for purification it is possible to estimate the fraction of cell surface protein by performing gel densitometry (see Materials and methods and figure legend) In line A 2.55 ± 1.2% and in line E 1.93 ± 0.38% of Kir6.2 is surface biotinylated (n ¼ 4, not a significant difference) Finally, our conclusion is further supported by the presence of substantial and comparable currents in line A and line E DISCUSSION In this study we report a comprehensive study in a mammalian cell line of the effects of deletion of the SUR1 C-terminus on assembly, trafficking and function of the KATP channel complex The data reported here show that the cytoplasmic C-terminus of SUR1 has a key role in channel function but is not absolutely required for complex assembly or trafficking Our data support aspects of previous studies but also extend those observations [11,13,14] The cytoplasmic C-terminus of SUR1 containing NBD2 is mutated in persistent hyperinsulinaemic hypoglycaemia of infancy The disease is autosomal recessive and is characterized by profound neonatal hypoglycaemia and Fig Analysis of channel subunit trafficking using surface biotinylation The Western blots shown in (A) and (B) were probed with : 2000 dilutions of anti-SUR1 and anti-Kir6.2 Ig, respectively Lanes labelled lysate were loaded with approximately 1.5% of the total lysate obtained from the surface biotinylated cells The lanes labelled AP were loaded with approximately a third of eluate obtained from the neutravidin binding column and corresponds to surface-biotinylated protein The blots shown represent exposures to film of 20–25 s The positions of molecular mass markers and their sizes in kDa are shown on the left of each blot The experiments were repeated on three further occasions with similar results In (A) it can be observed that coexpression of Kir6.2 + SUR1myc (line A) and Kir6.2 + SUR1del249myc (line E) results in increased surface biotinylated SUR1 protein compared with SUR1 expressed alone In (B), surface biotinylated Kir6.2 can be observed when coexpressed with either SUR1myc or SUR1del249myc Quantitative analysis was also performed to show that there was no difference in the proportion of surface biotinylated Kir6.2 when coexpressed with either SUR1myc or SUR1del249myc (refer to main text for more details) inappropriate insulin secretion from the pancreas A number of point mutations cluster in this region of SUR1; the resulting frameshift mutations lead to premature truncation of the protein [12] A number of studies have examined various aspects of the role of the C-terminus, including NBD2, in KATP channel function Sharma et al [11] performed limited deletions of the C-terminus and identified a forward trafficking signal that increased plasma membrane currents The removal of as few as seven amino acids led to loss of channels at the plasma membrane based on the reduction of sulfonylurea inhibited Rb+ flux, a chemiluminescent trafficking assay and the absence of an apparent higher molecular mass glycosylated species of SUR1 In contrast, Sakura et al [13] observed pronounced expression of plasma membrane currents that was not quantitatively different from wildtype In addition, Schwappach et al [14] showed that a Ó FEBS 2002 5312 J P Giblin et al (Eur J Biochem 269) SUR1-MRP1 C-terminal chimaera (in which the C-terminus of SUR1 was replaced by MRP1) when coexpressed with Kir6.2 could reach the plasma membrane One potential explanation for this is that the trafficking in these two systems is quite different Indeed it is now well established that the commonest of the mutations (DF508) in the CFTR protein (cystic fibrosis transmembrane conductance regulator) causing cystic fibrosis forms a functional chloride conductance in Xenopus laevis oocytes but not in mammalian cells The explanation is that the trafficking is temperature dependent: it occurs at 23 but not 37 °C [22,23] Furthermore, there are a number of rarer mutations in cystic fibrosis that affect mainly the C-terminus of CFTR and lead to premature truncation of the protein in an analogous fashion to those occurring in SUR1 in persistent hyperinsulinaemic hypoglycaemia of infancy In a series of elegant studies, Lukacs and colleagues have shown that trafficking through the secretory pathway is not disturbed and maturation occurs normally [24,25] However C-terminally deleted CFTR is degraded more quickly than wild-type protein via a novel transport mechanism that occurs from the plasma membrane to the proteosome [24,25] Our data in a human cell line support the idea that the deletion of the C-terminus of SUR1 does not give channel complexes predisposed to temperature dependent trafficking We also examined whether there was a subtle relative trafficking defect by using a quantitative immunofluorescence assay and cell surface biotinylation and were unable to demonstrate major disturbances in the trafficking However the assays may not be sensitive enough to pick up a decrease in forward trafficking from ER to Golgi or increase in degradation rate with C-terminal deletion as occurs with CFTR It is also conceivable, however, that these mutants may traffic differently in different cell lines and that there may be trafficking determinants in the region defined by the largest and smallest deletions It is worth pointing out that the immunofluoresence assay will not distinguish between protein at the membrane or in submembranous vesicles As might be expected given the ability of the SUR1 deletion mutants to form functional channels at the membrane, there was also no gross disturbance of interaction between the subunits as assessed by coimmunoprecipitation However, our data reveal quite profound disturbances in the function of the mutant channel complex The ATP sensitivity is not significantly disturbed and this is supported by more detailed studies in Xenopus laevis oocytes [13] More interestingly however, as observed by Sakura et al., profound deletion of the C-terminus of SUR1 generates channels that are insensitive to MgADP and diazoxide In addition we demonstrate here that only relative minor deletions also impair the ability of diazoxide to activate the channels In particular intact Walker A and B motifs are necessary for diazoxide activity The current models of the interrelationship of channel opener binding and action, ATP binding and hydrolysis and MgADP binding and stimulation are complex [19,20,26,27,27–34] Much of this data is compatible with the idea that the hydrolysis of MgATP at NBD2 results in an MgADP bound conformation that leads to channel activation Openers stabilize the latter state and this effect is modulated by nucleotide binding at NBD1 Our data emphasize the central role that a fully intact NBD2 of SUR1 plays in stimulation by MgADP and diazoxide and support the above hypothesis Sulfonylurea action is more subtly altered The Kd for tritiated glibenclamide binding is not significantly changed However tolbutamide, at doses known to interact predominantly with the sulfonylurea receptor [35], causes only a partial reduction in current but with an identical Ki Tolbutamide has the same affinity for interaction but the efficacy is decreased Our data differ from those of Sakura et al [13] who found no difference for high affinity tolbutamide block when measuring whole-cell currents in two-electrode voltage clamp recordings of macroscopic currents in Xenopus laevis oocytes (they did see some reduction in effect in inside-out patches) However, they used metabolic poisoning to elicit these currents and this may account for the difference In addition, we demonstrate that this phenomenon occurs with even relatively modest deletions What the observations say about the mechanism of sulfonylurea block? Recent studies [36,37] indicate that the binding pocket is located within the final group of transmembrane domains between NBD1 and NBD2 It is interesting that the coexpression of two SUR hemi molecules is necessary for the full reconstitution of the sulfonylurea binding site [38] Our data are compatible with the idea that the binding is unaltered but occupation of its site results in less pronounced effects and a change in efficacy dependent on NBD2 It emphasizes that the occupation of the sulfonylurea binding site need not necessarily translate itself into channel closure [39] Secondly it suggests that a significant proportion of the sulfonylurea effect may be directly related to the antagonism of MgADP induced openings dependent on an intact Walker A and B motif in NBD2 Speculatively, it supports an emerging picture in which NBD2 and the final group of transmembrane domains interact functionally to modulate the pore forming subunit Finally, these studies demonstrate as in our previous work [15] that assembly and trafficking may be well preserved but functional coupling between SUR1 and Kir6.2 is critically determined by a number of interdependent factors ACKNOWLEDGEMENTS This work was supported by BBSRC, British Heart Foundation, Diabetes UK and the Wellcome Trust We are grateful to Professor S Seino for providing Kir6.2 cDNAs and Professor J Bryan for providing SUR1 cDNA REFERENCES Seino, S (1999) ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies Annu Rev Physiol 61, 337–362 Babenko, A.P., Aguilar Bryan, L & Bryan, J (1998) A view of SUR/KIR6.X, KATP channels Annu Rev Physiol 60, 667–687 Tucker, S.J & Ashcroft, F.M (1998) A touching case of channel regulation: the ATP-sensitive K+ channel Curr Opin Neurobiol 8, 316–320 Isomoto, S., Kondo, C & Kurachi, Y (1997) Inwardly rectifying potassium channels: their molecular heterogeneity and function Jpn J Physiol 47, 11–39 Ho, K., Nichols, C.G., Lederer, W.J., 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assembly of beta-cell K (ATP) channels J Biol Chem 275, 3360–3364 Gribble, F.M., Tucker, S.J., Seino, S & Ashcroft, F.M (1998) Tissue specificity of sulfonylureas: studies on cloned cardiac and beta- cell K(ATP) channels Diabetes 47, 1412–1418  ... complex The data reported here show that the cytoplasmic C-terminus of SUR1 has a key role in channel function but is not absolutely required for complex assembly or trafficking Our data support aspects... DISCUSSION In this study we report a comprehensive study in a mammalian cell line of the effects of deletion of the SUR1 C-terminus on assembly, trafficking and function of the KATP channel complex. .. stimulation by MgADP and diazoxide and support the above hypothesis Sulfonylurea action is more subtly altered The Kd for tritiated glibenclamide binding is not significantly changed However tolbutamide,