Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis
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- Varadi, A., Grant, A., McCormack, M. et al. Diabetologia (2006) 49: 1567. doi:10.1007/s00125-006-0257-9
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ATP-sensitive K+ (KATP) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca2+ concentration or pH.
To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca2+ were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin.
SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca2+ uptake into mitochondria, and KATP channel regulators had no significant effect on intravesicular free Ca2+ concentrations or vesicular pH.
A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.
KeywordsBeta cell Calcium Insulin KATP Sulfonylurea Sulphonylurea
fluorescently labelled boron dipyrromethene difluoride
enhanced green fluorescent protein
ATP-sensitive potassium channel
inwardly rectifying K+ channel, 6.2
lysosome-associated membrane protein-1
large dense-core insulin-containing vesicle
mitochondrial glycerol phosphate dehydrogenase
sterol regulatory element binding protein-1c precursor
trans-Golgi network protein 38
Elevated glucose concentrations stimulate the secretion of insulin from pancreatic islet beta cells via enhanced metabolism of the sugar and increases in the intracellular ATP:ADP ratio [1, 2]. Closure of ATP-sensitive K+ (KATP) channels  then causes cell depolarisation, influx of Ca2+ and the release of stored insulin .
KATP channels exist as oligomeric structures comprising four copies each of a K+ channel of the inward rectifier class (Kir6.2), which form the channel pore, and four sulfonylurea receptor-1 (SUR1) subunits . Closure of KATP channels is pivotal to the actions of both nutrient secretagogues and sulfonylureas; changes in the activity of either subunit leads to defective insulin secretion and glucose homeostasis in rodents [5, 6, 7]. Moreover, mutations in either subunit are a common cause of hyperinsulinism in infancy [8, 9], whilst polymorphisms in the KCNJ11 gene, which generate a form (E23K) of Kir6.2 with decreased activity, are linked to type 2 diabetes in human populations [10, 11, 12]. Moreover, mutations in the KCNJ11 gene are responsible for ∼50% of cases of permanent neonatal diabetes mellitus [13, 14, 15].
Early work indicated that endogenous Kir6.2  and SUR1  are present on most, if not all, islet cell types in the mouse, but did not resolve the intracellular localisation of the channel. Recent studies  have suggested that a significant proportion of the total cellular content of Kir and SUR channels reside on intracellular structures. Studies using fluorescently labelled glibenclamide (a cell-permeant sulfonylurea) and antibody staining , demonstrated that KATP channel complexes are located predominantly on dense-core insulin-containing secretory vesicles, in line with earlier observations [19, 20], and may serve to mediate effects of sulfonylureas . However, Quesada et al.  reported binding of fluorescently labelled glibenclamide–boron dipyrromethene difluoride (glibenclamide-BODIPY-FL) principally to the nuclear envelope of primary beta cells.
Smith et al.  have proposed that mitochondrial KATP channels, reportedly present on mitochondria in both liver  and heart , may mediate some of the effects on sulfonylureas in beta cells. Acting via these channels, diazoxide may also reduce the mitochondrial membrane potential, and therefore ATP synthesis . However, Garlid has pointed out that it is unlikely that sufficient mitochondrial KATP channel activity is present to mediate such an effect .
A potential criticism of previous immunocytochemical investigations  has been uncertainty as to the identity of the bound antigen. Here, by combining complementary fractionation, immunopurification and immunocytochemical approaches, we have investigated the subcellular localisation of Kir6.2 and SUR1 in isolated islets and clonal MIN6 beta cells. The latter cells retain many of the properties of the parental mouse beta cells, including efficient synthesis and storage of insulin and glucose-stimulated insulin secretion .
We demonstrate the presence of the majority of cellular immunoreactive Kir6.2 and SUR1 on dense vesicles and the distal secretory pathway, and the absence of these channel subunits from mitochondrial membranes. However, arguing against an important role for vesicular KATP channels in intracellular ion homeostasis, Ca2+ uptake into neither organelle was affected by KATP channel regulators.
Materials and methods
Cell culture reagents were from GibcoBRL (Life Science Research, Paisley, UK) and molecular biologicals from Roche Diagnostics (Lewes, UK). Alexa Fluor goat anti-rabbit or anti-guinea pig 488 and 568 secondary antibodies were from Molecular Probes (Eugene, OR, USA). Guinea pig polyclonal anti-insulin antibody was from DAKO (Glostrup, Denmark). Guinea pig polyclonal anti-Kir6.2 antibody was raised against a peptide comprising the last 36 amino acids of Kir6.2 , and rabbit polyclonal anti-SUR antibodies against a peptide epitope comprising amino acids 625–650 of hamster SUR1 (unpurified serum used for western blotting) and against a peptide epitope comprising amino acids 743–760 of rat SUR1 (purified antibody used for immunoelectron microscopy). Rabbit polyclonal anti-glycerol phosphate dehydrogenase (mGPDH; mitochondrial marker) antibody was from Sigma (Poole, UK), and rabbit polyclonal anti-phogrin antibody (against amino acids 629–1003) was a kind gift from J. Hutton (Barbara Davis Center for Childhood Diabetes, Denver, CO, USA) . Rabbit polyclonal anti-trans-Golgi network protein 38 (TGN38)  and mouse monoclonal anti-human lysosome-associated membrane protein-1 (LAMP-1) specific antibodies were kindly provided by G. Banting (University of Bristol, Bristol, UK). Rabbit polyclonal anti-insulin receptor antibody was from Santa Cruz Biotechnology (Mile Elm, UK). Mouse monoclonal anti-sterol regulatory element binding protein-1c precursor (SREBP)  was from F. Foufelle (Unit 465 INSERM, Paris, France). Rabbit polyclonal anti-14-3-3-β antibody was raised against a peptide mapping at the amino terminus of the human protein (Autogen Bioclear UK, Mile Elm, UK). OptiPrep, a solution of iodixanol, was from Axis-Shield (Oslo, Norway).
Islet isolation and cell culture
Mouse islets were freshly isolated as described previously . MIN6 pancreatic beta cells (at passages 19–35) were cultured in DMEM supplemented with 15% (v/v) foetal calf serum (FCS), penicillin (100 U/ml), streptomycin (0.1 mg/ml), β-mercaptoethanol and l-glutamine (2 mmol/l) at 37°C in an atmosphere of humidified air (95%) and CO2 (5%) .
Subcellular fractionation using OptiPrep iso-osmotic density gradient centrifugation
Cells were homogenised in 0.3 mol/l sucrose, 1 mmol/l EDTA, 1 mmol/l MgSO4, 10 mmol/l 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH (pH 6.5); 1 μmol/l phenylmethylsulfonyl fluoride (PMSF), 5 μg/ml aprotinin, and 5 μg/ml leupeptin using a ball-bearing homogeniser, and then centrifuged at 500 g for 10 min. The post-nuclear supernatant was layered on top of a continuous 8–19% (w/v) OptiPrep gradient obtained using a Gradient Master (BioComp Instruments, Fredericton, NB, Canada) and centrifuged at 16, 000 g for 16 h. Gradient fractions were collected by downward displacement (Gradient station; BioComp Instruments).
Detection of Kir6.2 and SUR in subcellular fractions of MIN6 cells
Equal volumes from the gradient fractions were separated on 9% (w/v) polyacrylamide gels then blotted onto Immobilon-P transfer membrane (Millipore, Watford, UK) and probed with organelle-specific antibodies against mGPDH (mitochondria), phogrin (large dense-core insulin-containing vesicle [LDCV] membranes) [30, 35], TGN38 (Golgi) , insulin receptor (IR, plasma membrane); LAMP-1 (lysosomes) ; SREBP precursor (endoplasmic reticulum [ER]) ; 14-3-3-β (cytosol) . The protein and insulin contents in each fraction were determined using a Pierce BCA Protein Assay Kit (Rockford, IL, USA) and a Mercodia Ultrasensitive Mouse Insulin ELISA Kit (Uppsala, Sweden), respectively.
FACS sorting of phogrin.EGFP-containing vesicles and precipitation with trichloroacetic acid
MIN6 cells were infected with the recombinant phogrin.enhanced green fluorescent protein (EGFP) adenoviral construct, at a multiplicity of 30–100 viral particles/cell, for 1 h. Cells were subsequently used 24 h post infection when >95% of cells were infected. Cells were scraped into ice-cold buffer containing 10 mmol/l 3-(N-morpholino)propanesulfonic acid (MOPS), 260 mmol/l sucrose (pH 6.5), 1 mmol/l PMSF, 5 μg/ml aprotinin and 5 μg/ml leupeptin, then homogenised with a Teflon homogeniser and centrifuged at 500 g for 5 min. The post-nuclear supernatant was resuspended in MOPS buffer to a concentration of 1–2 mg/ml and sorted into two fractions: (1) particles labelled with EGFP, and (2) unlabelled organelles. Sorting was carried out on a FACS Vantage sorter (Becton Dickinson, Oxford, UK) fitted with a 488-nm argon ion laser. The EGFP fluorescence was measured using a bandpass filter at 530/30 nm. Following sorting, 7×106 vesicles were obtained, and 7×104 vesicles were seeded onto poly-l-lysine-coated coverslips and used for immunocytochemistry. The remaining vesicle suspension was treated with an equal volume of 20% (v/v) trichloroacetic acid for 30 min at 4°C, then centrifuged at 13, 000 g for 10 min before SDS gel electrophoresis.
FACS-sorted vesicles on glass coverslips were fixed for 10 min in 4% (w/v) paraformaldehyde in PBS at room temperature, followed by washing in PBS (5 min). Vesicles were incubated in 100 mmol/l glycine in PBS (pH 8.5) for 5 min and then 10% (v/v) FCS in PBS (5 min), before permeabilisation in 0.2% (v/v) Triton X-100 in PBS (20 min) at room temperature. This was followed by blocking in 3% (w/v) BSA in PBS for 15 min. Cells were then incubated with the primary antibodies overnight and with the secondary antibodies for 1.5 h at room temperature in 3% (w/v) BSA in PBS, before mounting and confocal imaging as described previously .
Images were captured on a Nipkov disc-based UltraVIEW confocal system (PerkinElmer Life Sciences, Boston, MA,USA)  using a 63× PL Apo 1.4NA oil-immersion objective lens (Leica, Heidelberg, Germany).
Immunoabsorption of phogrin
EGFP-containing vesicles. Phogrin.EGFP-infected cells were homogenised as described previously . The post-nuclear supernatant was further centrifuged at 2,400 g for 10 min at 4°C, and the pellet was resuspended in buffer B (50 mmol/l HEPES, 1 mmol/l EDTA, 150 mmol/l NaCl, 1 μmol/l PMSF, 5 μg/ml aprotinin and 5 μg/ml leupeptin) to a concentration of 1–2 mg/ml. A sample (100–200 μg) of homogenate was pre-cleared with 100 μl of packed Protein-A sepharose in buffer B overnight, and then centrifuged at 14,000 ×g for 3 s. Anti-EGFP antibody (20 μl) was first incubated with 50 μl Protein-A sepharose in buffer B. Pre-cleared samples (150–250 μl) were added to the antibody-bound beads, and incubation continued for a further 24 h at 4°C. Samples were centrifuged at 500 g for 30 s, and the immunoadsorbed vesicles were washed four times with buffer B and analysed by SDS-PAGE and immunoblotting . Immunostaining was revealed with horseradish peroxidase-conjugated anti-guinea pig IgG (1:5,000 dilution) and anti-rabbit IgG (1:40,000 dilution) using an enhanced chemiluminescence (ECL) detection system.
Postembedding immunocytochemistry and electron microscopy
Islets or MIN6 cell pellets were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/l sodium cacodylate buffer (pH 7.4) containing 3 μmol/l CaCl2 at 37°C, and allowed to cool to room temperature for 4 h. LR White resin-embedded ultrathin (70–150 nm) sections were picked up on uncoated nickel slot grids and were incubated on blocking solution, which consisted of 50 mmol/l Tris (pH 7.4) containing 0.9% NaCl (TBS), 0.1% Tween-20 and 2% BSA for 1 h. Sections were then incubated overnight at 4°C with immunopurified primary antibodies against insulin (25 μg/ml), Kir6.2 (5–50 μg/ml) and SUR1 (15–30 μg/ml). Sections were then incubated with goat anti-rabbit or goat anti-guinea pig IgG coupled to gold particles (10 nm diameter; 1:100; Nanopores, Stony Brook, NY, USA or BioCell International) for 2 h at room temperature. Selective labelling was not detected in the absence of primary antibodies. Cells were randomly chosen and photographed at 31,000× magnification using a Philips/FEI CM10 transmission electron microscope (Cambridge, UK).
Measurement of mitochondrial and vesicular-free Ca2+ concentration and pH
MIN6 cells were seeded onto glass coverslips coated with 13 mmol/l poly-l-lysine, and then grown to 50–80% confluence. Cells were infected  with adenoviruses encoding untargeted aequorin (AdCMVcytoAq) , aequorin targeted to the mitochondria (AdCMVmAq) , or mutant aequorin targeted to secretory vesicles (AdCMVVampAq) .
Aequorin was reconstituted with 5 μmol/l coelenterazine (LUX Biotechnology, Edinburgh, UK) by incubating for 2 h at 37°C in modified Krebs–Ringer bicarbonate buffer (KRB) (140 mmol/l NaCl, 3.5 mmol/l KCl, 0.5 mmol/l NaH2PO4, 0.5 mmol/l MgSO4, 3 mmol/l glucose, 10 mmol/l HEPES, 2 mmol/l NaHCO3, pH 7.4) supplemented with 1.5 mmol/l CaCl2. For reconstitution of VAMPAq, cells were depleted of Ca2+ by incubation with the Ca2+ ionophore ionomycin (10 μmol/l), the Na+/H+ exchanger monensin (10 μmol/l), and the SERCA inhibitor cyclopiazonic acid (10 μmol/l) in Ca2+-free KRB supplemented with 1 mmol/l EGTA, for 5 min at 4°C  prior to the addition of 5 μmol/l coelenterazine and further incubation for 2 h at 4°C in KRB supplemented with 100 μmol/l EGTA.
Intact cells were perifused at 37°C in a thermostatic chamber close to a photomultiplier tube (Thales UK, Addlestone, UK) . For experiments on permeabilised cells, an intracellular buffer (IB) was used (140 mmol/l KCl, 10 mmol/l NaCl, 1 mmol/l KH2PO4, 1 mmol/l ATP, 5.5 mmol/l glucose, 20 mmol/l HEPES and varying concentrations of MgSO4 and CaCl2, pH 7.05, plus 2 mmol/l Na+ succinate, unless otherwise stated. IB was buffered with 0.2 mmol/l EGTA and 1 mmol/l HEDTA, and the free [Ca2+] and [Mg2+] (usually 0.5 mmol/l) were calculated using ‘METLIG’ . MIN6 cells were initially perifused with KRB supplemented with 100 μmol/l EGTA before permeabilising with 20 μmol/l digitonin in IB for 1 min at 37°C. Aequorin calibration was as described previously [44, 48]. Vesicular pH was measured using ecliptic pHluorin and confocal microscopy .
Data are presented as the means±SEM for 3–6 separate experiments, and statistical significance calculated using the Student’s t-test.
Subcellular localisation of Kir6.2
To confirm the identity of the recognised epitope, we next used density gradients of iodixanol (OptiPrep)  to obtain subcellular factions of MIN6 cells. As shown in Electronic Supplementary Material (ESM) Fig. S1, this technique allowed separation of progressively heavier fractions from those rich in cytosol markers (14-3-3-β protein; fractions 1–5), early endosomes (LAMP-1; fractions 1, 2), ER (SREBP-1 precursor; fractions 2, 3), plasma membrane (insulin receptor; fractions 5 and 6) and Golgi apparatus (TGN38; fractions 8–11). LDCV markers, including phogrin (endogenous and overexpressed) and insulin, were principally found towards the bottom of the gradient, in fractions 9–13 (ESM Fig. S2), but were clear of mitochondrial contaminants (mGPDH, fraction 14).
Subcellular localisation of SUR1
Impact of KATP channel modulation on intravesicular-free Ca2+ and pH
Impact of KATP channel modulation on mitochondrial membrane potential and Ca2+ accumulation
Using complementary immunoelectron microscopy, cell fractionation and vesicle purification approaches, we show here that the subunits of the beta cell KATP channel are present chiefly on dense-core secretory vesicles but also more proximal regions of the secretory pathway in primary and clonal mouse beta cells. These data therefore refine a recent report  suggesting localisation of both subunits to LDCVs alone. In contrast, we failed to detect any immunoreactivity against either of the two classical beta cell KATP channel subunits on mitochondrial membranes (Figs. 1, 2, 3, 4, 5 and 6) or any pharmacological evidence for a role for these channels on beta cell mitochondria (Fig. 10). Whether the different intensity profiles of SUR1 and Kir6.2 (showing a greater enrichment of SUR1 on LDCVs; compare Figs. 1, 2 and 5) may be of functional significance is uncertain, but would be consistent with earlier studies . Thus, although we were unable here to demonstrate any role for KATP channels in intravesicular ion homeostasis, other roles for sulfonylurea binding to vesicles in regulating exocytosis, perhaps mediated by protein kinase C , are not excluded.
The subcellular localisation of SUR1, as revealed here using immunocytochemical techniques, is somewhat at variance with the findings of predominant nuclear envelope labelling with glibenclamide-BODIPY-FL in mouse beta cells , and might suggest the existence of a population of KATP channels at this site with a particularly high binding affinity for sulfonylureas. On the other hand, and in contrast to another report using this dye , we were able to demonstrate clear immunolabelling of non-dense-core vesicle intracellular membranes with anti-SUR1 antibodies, likely to correspond to ER/Golgi membranes, including the ER-contiguous nuclear envelope. These findings thus support a role for KATP channels in controlling Ca2+ release from the ER into the nucleus, and hence beta cell gene expression .
Might dense-core vesicles serve as sorting stations for the trafficking of KATP channels to the cell surface? This is an intriguing possibility, given that selective deposition of KATP channels at the plasma membrane during periods of continuous stimulation may contribute to the loss of glucose responsiveness observed in type 2 diabetes.
Supported by Wellcome Trust Programme Grant 067081/Z/02/Z. We thank M. Jepson and A. Leard (Bristol MRC Imaging Facility) for technical assistance, P. Cullen for the use of the UltraView confocal microscope, and A. Herman for FACS sorting. B. Schwappach and H. Yuan were supported by German Research Foundation Deutsche Forschungsgemeinschaft) Grant SFB638. G. A. Rutter is a Wellcome Trust Research Leave Fellow.
Duality of interest
The authors have no conflicts of interest.