The Insulin Secretory Granule Is the Major Site of KATP Channels of the Endocrine Pancreas Xuehui Geng,1 Lehong Li,1 Simon Watkins,1 Paul D

The Insulin Secretory Granule Is the Major Site of KATP Channels of the Endocrine Pancreas Xuehui Geng,1 Lehong Li,1 Simon Watkins,1 Paul D. Robbins,2 and Peter Drain1

an on/off switch. When on, potassium ions flow out With ATP sites on Kir6.2 that inhibit activity and ADP sites on SUR1 that antagonize the inhibition, ATP- through the channel electrically hyperpolarizing the ␤-cell, sensitive potassium channels (KATP channels) are de- putting a brake on the signal flow controlling insulin signed as exquisite sensors of adenine nucleotide levels secretion (11). When off, the KATP channel, which is that signal changes in glucose metabolism. If pancreatic inhibited by sulfonylureas or by the increased ATP/ADP KATP channels localize to the insulin secretory granule, ratio from glucose metabolism (12,13), removes this they would be well positioned to transduce changes in brake, allowing initiation of insulin release by elevating glucose metabolism into changes in granule transport intracellular calcium (14–17), which triggers the exocy- and exocytosis. Tests for pancreatic K channels lo- ATP totic fusion of insulin granule and ␤-cell membrane. calized to insulin secretory granules led to the following observations: fluorescent sulfonylureas that bind the The calcium signal, however, is insufficient for regulat- ing insulin release in response to glucose under typical pancreatic KATP channel specifically label intracellular punctate structures in cells of the endocrine pancreas. conditions (11,18). While pharmacologically either open- The fluorescent glibenclamides colocalize with Ins-C- ing or inhibiting the plasma membrane KATP channel, GFP, a live-cell fluorescent reporter of insulin granules. maneuvers that elevate not only intracellular calcium but Expression of either SUR1-GFP or Kir6.2-GFP fusion also glucose metabolism are necessary to confer glucose proteins, but not expression of GFP alone, directs GFP dose dependency to stimulated insulin release. By an fluorescence to insulin secretory granules. An SUR1 unknown mechanism, glucose metabolism provides a sec- antibody specifically labels insulin granules identified ond controlling parameter that, more distal in the path- by anti-insulin. Two different K 6.2 antibodies specifi- ir way, enhances the response of the insulin secretory cally label insulin secretory granules identified by anti- granule to the calcium signal, allowing amplification of insulin. Immunoelectron microscopy showed Kir6.2 antibodies specifically label perimeter membrane re- insulin release by elevated glucose metabolism. Study of gions of the secretory granule. Relatively little or no the amplification pathway has implicated adenine nucleo- labeling of other structures, including the plasma mem- tides as one of the factors coupling glucose metabolic and brane, was found. Our results demonstrate that the insulin exocytotic rates (16,18,19). Designed for ATP and insulin secretory granule is the major site of KATP ADP sensing, KATP channels could confer graded glucose channels of the endocrine pancreas. Diabetes 52: dose dependency in the amplification pathway in addition 767–776, 2003 to their role as on/off switch at the plasma membrane, elevating intracellular calcium. Because the response of the insulin secretory granule is involved, the distal cellular major question in insulin secretion is the site for the KATP channels might be the secretory granule. cellular site of action of sulfonylureas, which In this study, we used novel live cell confocal imaging, as are taken daily by millions of diabetic subjects well as classical immunohistochemical techniques, to test to correct hyperglycemia. One site of sulfonyl- for pancreatic KATP channels localized to insulin secretory A granules. We report on numerous lines of evidence indi- urea action is at the cytoplasmic face of the plasma membrane sulfonylurea receptor (1–5) of the ATP-sensi- cating not only that pancreatic KATP channels localize to tive potassium channel (K channel) (6). Hundreds of the secretory granule membrane but that it is their major ATP site in the ␤-cell of the endocrine pancreas. KATP channels are localized to the plasma membrane of ␤ insulin-secreting -cells (7). The pancreatic KATP channel comprises four regulatory sulfonylurea receptor (SUR1) RESEARCH DESIGN AND METHODS subunits and four potassium pore-forming (Kir6.2) sub- Islet isolation, infection, and culture. Murine islets were isolated from units (8–10). The plasma membrane KATP channel acts like BALB/c or C57BL/6 mice pancreata by collagenase digestion followed by separation on a Ficoll gradient and purified by hand under a stereo microscope (20). Recombinant Ad.Ins-C-GFP virus was used as described for islet From the 1Department of Cell Biology and Physiology, University of Pitts- infection (21). Islets were washed using serum-free culture medium and then burgh School of Medicine, Pittsburgh, Pennsylvania; and the 2Department of the medium was nearly completely removed by pipeting, leaving ϳ50 ␮l. The Molecular Genetics and Biochemistry, University of Pittsburgh School of expression levels shown were typical after 1–3 days of infection. Native and Medicine, Pittsburgh, Pennsylvania. infected islets were cultured in RPMI-1640 media containing 7.5 mmol/l Address correspondence and reprint requests to Peter Drain, Biomedical glucose and 10% FCS and incubated at 37°C in 5% CO . Live cells were used Science Tower South, Room 323, 3500 Terrace St., Pittsburgh, PA 15261. 2 for the experiments reported here, as determined by the LIVE/DEAD Viability/ E-mail: [email protected]. Received for publication 30 August 2002 and accepted in revised form 10 Cytotoxicity Kit (Molecular Probes, Eugene, OR), granule exocytotic capacity, December 2002. or both. Fluorescent sulfonylurea labeling. gKATP, granule ATP-sensitive potassium channel; KATP channel, ATP-sensi- Green glibenclamide BODIPY FL and tive potassium channel; KD, apparent dissociation constant. red glibenclamide BODIPY TR (Molecular Probes) were each used to achieve

DIABETES, VOL. 52, MARCH 2003 767 INSULIN SECRETORY GRANULE KATP CHANNEL

a final concentration of 40 nmol/l (22). Islets were washed using Krebs-Ringer buffer (in 7.5 mmol/l glucose) and then labeled by superfusion with the fluorescent glibenclamide within 60 min at 4°C until cellular fluorescence was readily detectable. The islets were then washed twice with Krebs-Ringer buffer and imaged. Confocal fluorescence microscopy. Islets were placed into an optical recording chamber (Harvard Apparatus, Holliston, MA) at 37°C. Single-photon confocal microscopy was performed using an Olympus Fluoview 300 Confocal Laser Scanning head with an Olympus IX70 inverted microscope (Olympus, Melville, NY). Excitation of GFP, green glibenclamide BODIPY FL, or green AlexaFluor 488 conjugated to secondary antibody was by the 488 nm Argon laser line and emission detected using sharp cutoff 510IF long-pass and BA530RIF short-pass filters for PMT 1. Excitation of red glibenclamide BODIPY TR or red AlexaFluor 594 conjugated to secondary antibody was by the 543-nm green HeNe laser line and emission detected using a sharp cutoff BA610IF long-pass filter for PMT 2. Coimaging was done by sequential excitation, and simultaneous detection of emission which showed no cross- talk was detectable under these excitation-detection conditions. DNA constructs and expression Green fluorescent protein within C-peptide of mouse insulin. Live cell insulin granule fluorescent labeling with the Adlox recombinant, designated simply “Adlox.Ins-C-GFP,” was used as described (21). Emerald GFP fused separately to COOH-termini of Mouse SUR1 and

Kir6.2. Overlap PCR was used to insert in-frame immediately after the coding

sequence of mouse SUR1 or mouse Kir6.2 in pCS2 (23), with an eight-glycine codon linker between coding segments. The simian CMV promoter of pCS2 ␮ was used to express SUR1-GFP or Kir6.2-GFP fusions by incubating 4 g DNA in 200 ␮l OptiMem media (Gibco/BRL, Gaithersburg, MD) in one tube and 8 ␮l Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 200 ␮l OptiMem media in a separate tube for 5 min at room temperature. The tubes were then combined, incubated for 20 min at room temperature, and added to islets.

KATP channel antibodies and confocal immunofluorescence microscopy. We raised rabbit anti-mouse SUR1 antibodies against the 21 COOH- terminal amino acid residues of mouse SUR1 (KPEKLLSQKDSVFASFVRADK [24]), and they were affinity purified with the peptide. We raised rabbit

anti-mouse Kir6.2 antibodies against either mouse Kir6.2 residues 377–390 (14 COOH-terminal residues KAKPKFSISPDSL [25]) or Kir6.2 residues 329–344 (DYSKFGNTIKVPTPLC), and each afﬁnity puriﬁed by using the corresponding

peptide. The SUR1 and Kir6.2 antibodies detected bands of the predicted molecular weights by Western analysis, and their Western and immunohistochemical signals were blocked by the cognate peptides used to generate the antibodies. Guinea pig anti-insulin was obtained from Dako (Carpinteria, CA). AlexaFluor 594 goat anti-rabbit IgG(HϩL) and AlexaFluor 488 anti-guinea pig IgG(HϩL) were from Molecular Probes. Freshly purified islets, naı¨ve or infected by Ad.Ins-C-GFP for 24–48 h, were fixed with 2% paraformaldehyde in PBS for 20 min, washed in PBS three times, and blocked by incubation in 2% BSA in PBS (pH 7.5) overnight at 4°C. The islets were then incubated with primary antibodies (as indicated, each at 5 ␮g/ml) in blocking buffer overnight at 4°C. The islets were washed three times with PBS, incubated with labeled secondary antibodies in blocking buffer for2hatroom temperature, and then washed three times with PBS. Confocal microscopy to detect the immunofluorescence was as described above. Immunoelectron microscopy. Islets were prepared for electron microscopy as described (21). Briefly, islets were cryofixed, cut into ultrathin (70–100 nm) sections with a Reichert Ultracut U ultramicrotome/FC4S cryo-attachment, and then lifted on a 2.3 mol/l sucrose droplet on Formvar-coated copper grids. Sections were washed with PBS and PBG buffer (PBS, 0.5% BSA, and 0.15% glycine), blocked with 5% normal goat serum PBG (30 min), labeled with

rabbit anti-Kir6.2 “COOH-terminus” at 1:100 in PBG for 1 h, washed in PBG, labeled with goat anti-rabbit gold-conjugated secondary antibodies (each at a FIG. 1. Fluorescent sulfonylureas bind punctate structures within cells of the endocrine pancreas. A: Green glibenclamide BODIPY FL labels dilution of 1:25 for 1 h), washed in PBG and PBS, fixed in 2.5% glutaraldehyde/ subcellular cytoplasmic structures that resemble secretory granules PBS, washed in PBS, and then washed in double-distilled H2O. Sections were within a single islet cell. Z sections (left to right and down) of a single poststained in 2% neutral uranyl acetate (7 min), washed in double-distilled isolated cell were taken every 0.5 ␮m. B: Red glibenclamide BODIPY TR H2O, stained in 4% uranyl acetate (2 min), and then embedded in 1.25% labels similar granule-like structures within a pair of islet cells. Every methylcellulose. Labeling was observed on a JEOL JEM 1210 electron 0.5 ␮m (left to right and down), z sections are shown of a pair of microscope (Tokyo) at 80 kV. isolated cells. The plasma membrane show no detectable fluorescence after labeling by either the green or red fluorescent sulfonylurea. Each was used at 40 nmol/l on freshly isolated live cells gently dissociated from mouse islets in RPMI-1640 culture medium with 7.5 mmol/l RESULTS glucose. PlanApo, 60X oil, NA 1.4. Bar equals 2 ␮m. Glibenclamide, a potent insulin secretagogue, binds granule-like structures in islet cells. While covisualiz- We found that either green fluorescent glibenclamide ing the arrival of secretagogue with release of insulin BODIPY FL or red fluorescent glibenclamide BODIPY TR granules, we were struck by the observation that the at 40 nmol/l applied to freshly isolated mouse islet cells sulfonylurea secretagogue glibenclamide, at low concen- localized to punctate cytoplasmic structures with spatial trations, localized strongly to the secretory granule itself. and dynamic properties reminiscent of insulin secretory

768 DIABETES, VOL. 52, MARCH 2003 X. GENG AND ASSOCIATES granules (Fig. 1). At up to 10-fold higher concentration, the fluorescent BODIPY dyes by themselves did not localize to subcellular structures, indicating that the glibenclamide moiety directed the fluorescent moieties to the puncta. Furthermore, application of unlabeled 4,000 nmol/l glibenclamide largely prevented labeling of the granules by 40 nmol/l fluorescent glibenclamides. The results suggest high-affinity sulfonylurea receptors localized to the insulin secretory granule, consistent with previous findings (26,27). Islet glibenclamide-labeled puncta are insulin secretory vesicles. We demonstrated that the major receptor site for the glibenclamides was localized to insulin secretory granules by covisualizing red glibenclamide together with the green fluorescent “Ins-C-GFP” reporter of insulin granules (21) expressed in the same islets. Application of 40 nmol/l red glibenclamide onto islets expressing Ins-C- GFP resulted in colocalization of the red and green fluorescent reporters at the same cytoplasmic punctate structures (Fig. 2). As before, the red glibenclamide bound to subcellular puncta. Green Ins-C-GFP fluorescence expressed in some of the ␤-cells of the same islet colocalized with red glibenclamide patterns. We studied this more closely at the single-cell level (Fig. 3). Insulin secretory granules identified by green Ins-C-GFP overlapped exten- sively with the red fluorescent glibenclamide. Obvious colocalization observed as yellow fluorescence was found by optically sectioning throughout the ␤-cell. Furthermore, the fluorescent glibenclamide resulted in high-intensity signals at the granule relative to other cell structures, suggesting that a substantial number of high-affinity sulfonylurea receptors are natively expressed at the insulin secretory granule. Little or no labeling of the ␤-cell membrane, or any other membranes than those of the secretory granules, was detected. The green glibenclamide had been previously shown to bind SUR of bovine monocytes and muscle cells with a subnanomolar apparent dissociation ϳ constant (KD) 40 nmol/l (22). SUR1 is likely the only receptor of the endocrine pancreas competent to appreciably bind 40-nmol/l fluorescently labeled glibenclamides. The results suggest that the high-affinity sulfonylurea receptor at the insulin granule is SUR1 (previously shown to be a subunit of the pancreatic KATP channel), which also resides on the ␤-cell plasma membrane (1–7,12,13). Expressed SUR1-GFP trafficks to insulin secretory granules. The results provide strong evidence for high- affinity sulfonylurea receptors localized to insulin secretory granules but do not directly identify the receptor as SUR1. The high intensity of the fluorescent glibenclamide signals at the insulin granule suggests fluorescent tags directly on SUR1 should be detectable. To test this, we FIG. 2. The insulin granule reporter green Ins-C-GFP colocalizes with determined whether SUR1 tagged with GFP localizes red fluorescent glibenclamide receptor sites within cells of the endo- green fluorescence to insulin granules. We used the CMV crine pancreas. A: Cells within an islet infected with Ad.Ins-C-GFP promoter to express a COOH-terminal GFP fusion to the after 2 days of expression. A subset of cells within the islet shows green fluorescent insulin granules containing green fluorescent Ins-C- mouse SUR1 (“SUR1-GFP”) subunit of the mouse pancre- GFP. The subset is due in part to adenovirus typically infecting a atic KATP channel (23). We determined whether islet cells minority of islets cells and in part to the Ins-C-GFP transcribed by the expressing green SUR1-GFP puncta could be colabeled insulin II gene promoter expressing only in insulin-secreting ␤-cells, which are ϳ60% of the total islet cells. B: Glibenclamide BODIPY TR with the red glibenclamide. Figure 4 demonstrates the (red fluorescence). C: Merge of A and B. Note perimeter labeling colabeling at granule-like structures observed. Small and reflects labeled granules lined up along cell membrane. Plasma membrane labeling would be at the diffraction limit, resulting in far thinner, large size classes of punctate structures were labeled by perfectly uniform fluorescent lines that would not exhibit variable SUR1-GFP and red glibenclamide. The small granules thickness. PlanApo, 60X oil, NA 1.4. Bar equals 2 ␮m. exhibited more uniform diameters averaging ϳ400 nm and

DIABETES, VOL. 52, MARCH 2003 769 INSULIN SECRETORY GRANULE KATP CHANNEL

FIG. 3. Single-cell detail of Ins-C-GFP colocalized with red fluorescent glibenclamide. A: Green Ins-C-GFP identifies insulin secretory granules of a cell within an islet. B: Glibenclamide BODIPY TR (red fluorescence) localization to puncta. Most of the red glibenclamide overlaps at punctate structures with the green Ins-C-GFP, but there are green granules that are not also red and there are red structures without green Ins-C-GFP. C. Merge of the red and green images; yellow intensity indicates punctate structures colocalizing red glibenclamide and the green insulin secretory granule reporter Ins-C-GFP. D: Optical sections, 2 ␮m apart, including the section exploded in the previous panels. PlanApo, 60X oil, NA 1.4. are bona fide insulin secretory granules, consistent with The SUR1-GFP results, in addition to the fluorescent previous measurements (21). Colocalization of green glibenclamide results, suggest that native SUR1 might be SUR1-GFP and red fluorescent glibenclamide at secretory highly trafficked to insulin granules. granules was evident by the small yellow puncta in merged Native SUR1 localizes to insulin secretory granules images. Little or no fluorescence was detectable at the cell by immunofluorescence. To directly test for native SUR1 perimeter, consistent with a dramatically lower density of localized to insulin secretory granules, we took an immu- KATP channel protein at the plasma membrane (7). The nohistochemical approach. Antibodies against a COOH- large punctate structures strictly correlated with long-term terminal peptide of mouse SUR1 were used with anti- culturing, which was done to achieve substantial expres- insulin antibodies to probe freshly purified mouse islets. sion of the fluorescent KATP channel subunit-GFP report- Figure 5 shows extensive colocalization of the anti-SUR1 ers. Large puncta were found in naive islets, as well as and anti-insulin antibodies at secretory granules. Colocal- islets expressing transgenes, and were detectable by Ins- ization is evident by extensive yellow patterns of fluores- C-GFP or anti-insulin, indicating a close relationship to cence in the merged image (n ϭ 34 islets in seven insulin granules. The large puncta might plausibly be lysosomal intermediates containing insulin-dense cores experiments). Note that in these islets freshly prepared (28) or nascent insulin granules that failed to mature (29). from the animal, only the small uniformly sized puncta Further study of the large puncta was obviated by their that best approximate insulin granules are observed. Pre- absence in fresh islets where the anti-insulin and anti– incubation with the peptide that generated the anti-SUR1 antibodies, or omission of the SUR1 antibodies, resulted in KATP channel subunit antibodies always identified more characteristic, small-sized insulin secretory granules (see no detectable fluorescence. The results suggest native below). Previously, we had demonstrated that the GFP SUR1 are highly localized to insulin secretory granules, as used in the construction of SUR1-GFP, expressed alone, compared with the ␤-cell membrane. Because the SUR1 resulted in uniform green fluorescence in the cytoplasm, subunit has thus far always associated with the pore- which accumulates in the nucleus (21). Clearly, SUR1 forming subunit Kir6.2 in the KATP channel (6,8–10), the protein sequences directed the GFP fluorescent moiety not results might be further supported by testing for a granule to these compartments but to insulin secretory granules. site for Kir6.2.

770 DIABETES, VOL. 52, MARCH 2003 X. GENG AND ASSOCIATES

FIG. 4. Exogenously expressed SUR1-GFP colocalizes with red fluorescent glibenclamide at insulin secretory granules. A: Localization of SUR1-GFP (green fluorescence) to punctate structures of a cell within an islet. B: Localization of acutely applied glibenclamide BODIPY TR (red fluorescence). C: Merge of green SUR1-GFP and red glibenclamide. Yellow intensity indicates punctate structures colocalizing the green SUR1-GFP and the red glibenclamide. Images were collected sequentially with 488-nm line excitation followed by 543-nm line excitation and then merged offline. D: Optical sections, 2 ␮m apart. Note that large puncta were detected by SUR1-GFP only after long-term culturing needed to obtain substantial fluorescent signal. The large puncta were also labeled consequent to long-term culturing of naı¨ve as well as transgenic islets by anti-insulin antibodies, indicating a close relationship with insulin granules. The small puncta best approximate insulin granule size. PlanApo 60X oil, NA 1.4.

Expressed Kir6.2-GFP trafficks to secretory granules. the COOH-terminal cytoplasmic domain of mouse Kir6.2 We tested for targeting to granules of expressed Kir6.2- and colabeling by anti-insulin. Figure 7 shows that native GFP fusions of the mouse pancreatic KATP channel (23). Kir6.2 is localized to the insulin secretory granule. An Figure 6 shows that simultaneous expression of green antibody to the C-terminus of Kir6.2 directed red fluores- fluorescent Kir6.2-GFP and labeling by 40 nmol/l red glib- cent secondary antibody to insulin granules, identified by enclamide results in colabeling indicated by yellow puncta the anti-insulin labeled by green fluorescent secondary ϭ in islet cells. Extensive colabeling was observed through- antibody (n 24 islets in five experiments). Native Kir6.2 out optical sections in all cells (n ϭ 47 islets in five in the ␤-cell membrane was not detectable under these experiments). The average fluorescence diameter was conditions, in which granule Kir6.2 protein labeling was 0.435 Ϯ 0.231 ␮m(n ϭ 283 granules in seven cells of five strong. A second antibody directed against another pep- islets in three experiments). A minority of granules tide of the COOH-terminal cytoplasmic domain of Kir6.2 showed one but not the other label, consistent with high showed highly similar results (data not shown). The Kir6.2 Kir6.2-GFP expression and low diffusion of red gliben- peptides used to generate the antibodies prevented all clamide, or low Kir6.2-GFP expression and complete dif- labeling by their respective antibodies, as did omission of fusion. No plasma membrane fluorescence was detectable. the primary antibodies. Given that Kir6.2 is an integral As mentioned, exogenously expressed GFP alone is not membrane protein, antibodies against Kir6.2 might label targeted to the insulin granule (21). Localization to secre- specifically at the membrane perimeter of the secretory tory granule of pore-forming Kir6.2-GFP fusions, but not granule. GFP alone, suggests that native KATP channel subunits Native Kir6.2 resides in the membrane of the secre- traffick to insulin secretory granules. tory granule. For the immunoelectron microscopy exper- Native Kir6.2 localizes to insulin secretory granules. iments, we prepared 70- to 100-nm thin sections of mouse We tested for native Kir6.2 localized to the granule by islets. Figure 8 shows that gold particle–conjugated sec- labeling with either of two distinct antibodies specificto ondary antibodies against anti-Kir6.2 COOH-terminus anti-

DIABETES, VOL. 52, MARCH 2003 771 INSULIN SECRETORY GRANULE KATP CHANNEL

experiments). The gold particle labeling was associated with the membrane of the secretory granule and not the dense core. The results show that native Kir6.2 subunits are largely localized to the membranes of insulin secretory granules in ␤-cells.

DISCUSSION The main finding of this study is the demonstration by a variety of approaches that the major site for the pancreatic KATP channel is the insulin secretory granule. That the fluorescent glibenclamides labeled native sulfonylurea receptors localized to insulin secretory granules was further supported by the observation that red fluorescent glibenclamide colocalized with the insulin fluorescent reporter, Ins-C-GFP. We have shown that Ins-C-GFP labels secretory granules that can be mobilized and exocytosed by insulin secretagogues (21). Our results corroborate previous evidence for a sulfonylurea receptor at the insulin secretory granule. For example, early work by Hellman et al. (30) showed evidence for dramatic uptake of glibenclamide into islets. Another study combined 3H- glibenclamide labeling, autoradiography, and electron microscopy to determine an insulin granule receptor for the sulfonylurea (26). We extended our results by using a quite different second approach. Expression in islet ␤-cells of the pancreatic high-affinity sulfonylurea receptor fused to GFP (SUR1-GFP), but not the GFP expressed alone, localized fluorescence to the same intracellular punctate structures labeled by red fluorescent glibenclamide. Anti-SUR1 antibody immunohistochemistry was a third approach that indicated insulin secretory granules as the major site for SUR1. The three results each support the conclusion that native SUR1 is targeted to insulin secretory granules. Previous observations are consistent with our findings. The pancreatic SUR1 protein has an apparent molecular weight of 140 kDa with more highly glycosylated forms occurring (8). Ozanne et al. (27) showed, by density gradient centrifugation and 3H-glibenclamide labeling, that Ͼ90% of the glibenclamide mapped to intracellular structures, with the majority associated with insulin secretory granules. They showed evidence for 3H-glibenclamide binding to intracellular receptors with nanomolar affinity and for 3H-glibenclamide cross-linking to 140- and 170-kDa proteins. Additional results implicate a sulfonylurea receptor protein of 65-kDa in zymogen as well as insulin granules (31), which might have also contributed to the granule fluorescence reported here. A secretory granule SUR1 suggested its Kir6.2 counter- part in the pancreatic KATP channel would also be targeted to secretory granules. We showed that expressed Kir6.2- FIG. 5. Anti-SUR1 peptide antibodies localize at secretory granules GFP fusions localized to insulin granules labeled by red identified by anti-insulin. A: Anti-insulin antibodies (green fluores- fluorescent glibenclamide. Second, two different anti- cence) identifiy secretory granules in a ␤-cell gently dissociated from freshly purified islets. B: Anti-SUR1 peptide antibodies identify similar Kir6.2 antibodies indicated insulin secretory granules as ␤ pattern of punctate structures in the same cell. C: Merge of green the major -cell site for native Kir6.2. Third, immunoelec- insulin secretory granule labeling and red SUR1 labeling. PlanApo, 60X oil, NA 1.4. tron microscopy with the anti-Kir6.2 antibodies demonstrated a membrane location of dense-core granules for Kir6.2. Taken together, our results provide the first evi- bodies localized around dense cores, decorating the dence specifically identifying SUR1 and Kir6.2 localized to perimeter of secretory granules, while little or none insulin secretory granules. Given that SUR1 and Kir6.2 Յ ( 0.02) localized to plasma membrane, mitochondria, assemble into KATP channels in the endoplasmic reticulum ϭ nucleus, or other cellular structures (n 11 islets in three (32–34), we propose that pancreatic KATP channels are

772 DIABETES, VOL. 52, MARCH 2003 X. GENG AND ASSOCIATES

FIG. 6. Exogenously expressed Kir6.2-GFP trafficking to secretory granules colabeled by red glibenclamide BODIPY TR. A: Localization of Kir6.2-GFP (green fluorescence) labels punctate structures in a cell within an islet. B: Localization of acutely applied glibenclamide BODIPY TR (red fluorescence) to punctate structures within the same cell. C: Merge of previous images where yellow reflects colocalization. D: Optical sections, 2 ␮m apart, including the section exploded in the previous panels. The large puncta are associated with long-term culture, which was routinely done to obtain significant punctate fluorescence from Kir6.2-GFP. PlanApo, 60X oil, NA 1.4. then targeted by unknown mechanisms to insulin secre- ureas decreases potassium permeability, leading to Ca2ϩ tory granules. Once inserted into the ER membrane, the influx and insulin release, seemed reasonable at the time KATP channel, like other proteins, is expected to maintain and explains much of what we currently know of first- its membrane topology for all cellular membranes. Thus, phase insulin release. Like most studies on the action of the SUR1 COOH-terminus and Kir6.2 NH2- and COOH- sulfonylureas on insulin secretion, these studies fail to termini are cytoplasmic at granule as well as plasma address any other cellular domain but the plasma mem- membranes. brane. Yet the experimental results are nearly always Implications of granule KATP channels of the endo- interpreted to mean that the isulinotropic action of sulfo- crine pancreas. Nearly all studies on glucose-stimulated nylurea compounds is exclusively at the plasma mem- insulin secretion signaling cite solely a plasma membrane brane, and not internalized. Our results indicate that this ␤ -cell KATP channel. Early on, Gylfe et al. (35) reviewed view is incomplete and cellular mechanisms of pancreatic numerous published data on islet uptake characteristics of KATP channels might be productively studied at the in- radioactive sulfonylurea compounds. The five sulfonylurea sulin secretory granule membrane, as well as the plasma compounds studied, including tolbutamide, showed rapid membrane. saturated binding with little uptake, suggesting interac- Whenever we observed intense fluorescence localized to tions with the surface of the ␤-cells. Glibenclamide, how- secretory granules, little if any cell membrane labeling was ever, did not rapidly reach uptake equilibrium, but rather detected. The relative density of the pancreatic KATP progressively accumulated in substantial quantity within channel in the secretory granule membrane is likely high the islets (30). While discussing this exceptional mode of relative to the ␤-cell membrane. The observation is con- interaction of glibenclamide, Gylfe et al. nevertheless sistent with electrophysiological estimates of the plasma concluded that the insulin-releasing action of these drugs membrane KATP channel, where hundreds are thought to ␤ is due to binding with the -cell membrane, because most reside (7). On one hand, the KATP channels spread across of the sulfonylurea compounds were not appreciably the ␤-cell plasma membrane would be largely undetect- internalized yet act as insulin secretagogues. The overall able by the confocal microscopy conditions used here, but model presented, that cell-surface interaction of sulfonyl- are readily detectable by electrophysiology. On the other

DIABETES, VOL. 52, MARCH 2003 773 INSULIN SECRETORY GRANULE KATP CHANNEL

FIG. 7. Anti-Kir6.2 peptide antibodies localize at secretory granules identified by anti-insulin. A: Anti-insulin antibodies (green fluorescence) ␤ labeling in a cluster of -cells within a freshly purified islet. B: Anti-Kir6.2 peptide antibodies (red fluorescence) applied to the same cells. C: Merge of the green insulin and red Kir6.2 images where yellow shows areas of obvious colocalization of insulin granules and Kir6.2. D: Localization ␤ of insulin secretory granules by anti-insulin (green fluorescence) in a -cell dissociated from a freshly purified islet. E: Localization of anti-Kir6.2 antibodies (red fluorescence) in same ␤-cell. F. Merge of previous images where yellow shows obvious colocalization of green insulin and red

Kir6.2 labeling. PlanApo, 60X oil, NA 1.4. ␤ hand, the electrophysiology that detects KATP channels at exceptional ability to be internalized within -cells (30,35), the cell membrane is incapable of testing for KATP chan- but also by its superior secretory efﬁcacy (3). The obser- nels at granule membranes. Interestingly, glibenclamide is vations call for sulfonylurea drug design in the future to distinguished from other sulfonylureas, not only by its speciﬁcally target the intracellular secretory granule sites.

774 DIABETES, VOL. 52, MARCH 2003 X. GENG AND ASSOCIATES

FIG. 8. Anti-Kir6.2 antibodies localize to secretory granule perimeters. A: Immunoelectron microscopy of dense core secretory granules with anti-Kir6.2 peptide antibodies labeled with gold particle–conjugated secondary antibody. Mitochondrion in upper left and other structures did not localize gold particles. B: Independent experiment also showing anti-Kir6.2 labeling of dense-core secretory granules. Note frequent separation of gold particle labels from dense core, consistent with secretory granule membrane localization of Kir6.2. Cells imaged were from thin sections .of intact islets. Scale bar is 200 nm. Magniﬁcation is ؋30,000

Our results suggest there are two roles for pancreatic phospholipid, and protein interactions between the two KATP channels that depend on cellular position. KATP membrane domains. Thus, via increases in ATP and de- channels localized to the ␤-cell membrane play a well- creases in ADP that combine with changes in intracellular established role in coupling glucose metabolism to plasma calcium and phosphoinositides, glucose metabolism could membrane excitability that switches calcium influx on for alter gKATP channel activities, involving interactions with release. The calcium influx triggers exocytosis of primed lipids, motors, and associated proteins that mediate trans- insulin granules docked at the ␤ plasma membrane, ob- port, recruitment, and exocytosis of the granule at the ␤ served as first-phase release (36). KATP channels localized -cell membrane (44–50). Whereas the minority of KATP to the insulin granule membrane might also play a role in channels in the plasma membrane provide on/off switch coupling glucose metabolism in a dose-dependent manner regulation for Ca2ϩ influx that mediates first-phase release to granule transport and exocytosis, observed as second- and is permissive for second-phase release (11,14–18), the phase release (see below). Transgenic studies show that majority of KATP channels in secretory granule membranes inactivation of the pore-forming Kir6.2 subunit renders are situated to accelerate, in a graded way with glucose glucose ineffective on insulin secretion from isolated is- metabolism, granule translocation, priming, and exocyto- lets, with a very small first-phase possibly remaining (37). sis rates that otherwise limit second-phase insulin release. Transgenic inactivation of SUR1 also disrupts the response of both phases to glucose, where only a dramatically delayed and blunted release was found that might or ACKNOWLEDGMENTS might not be mechanistically related to wild-type second- This study was supported by the Department of Cell phase release (38). Even with calcium chronically elevated Biology and Physiology of University of Pittsburgh School by the SUR1 inactivation, second- as well as first-phase of Medicine, the Juvenile Diabetes Research Foundation response to elevated glucose was disrupted, consistent (grant no. 4-1999-845), and the National Science Founda- with granule KATP (gKATP) channels mediating an amplify- tion (grant no. MCB 9817116). ing action of glucose on insulin release. The authors thank Drs. Meir Aridor, Bob Bridges, Ray gKATP channels are designed and positioned to confer Frizzell, Linton Traub, and Massimo Trucco for helpful glucose dose dependency to the second phase of insulin discussions, and Rita Bottino, Bala Balamurugan, and release, where calcium is unlikely rate limiting (14,16, Yigang Chen for generously providing highly pure mouse 39,40). There is evidence that the ATP/ADP ratio exerts islets. This study is dedicated to the memory of Paul F. control strength over signal flow, coupling glucose metab- Drain, who taught us measure and patience. olism and insulin exocytosis rates. In particular, increases in ATP/ADP ratios that signal elevated blood glucose levels accelerate steps distal to calcium influx through the REFERENCES plasma membrane, including insulin granule translocation 1. Schwanstecher M, Schwanstecher C, Dickel C, Chudziak F, Moshiri A, Panten U: Location of the sulphonylurea receptor at the cytoplasmic face and exocytosis (16,18,19,41). With ATP sites on Kir6.2 that of the ␤-cell membrane. Br J Pharmacol 113:903–911, 1994 inhibit activity and ADP sites on SUR1 that antagonize the 2. Aguilar-Bryan L, Nichols CG, Weschler SW, Clement IV JP, Boyd III AE, inhibition, gKATP channels are designed as ATP and ADP Gonzalez G, Herrera-Sosa H, Nguy K, Bryan J, Nelson DA: Cloning of the sensors of glucose metabolism. The increased ATP/ADP ␤ cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. ratio could inhibit granule membrane potassium conduc- Science 268:423–426, 1995 3. Ashcroft FM: Adenosine 5Ј-triphosphate-sensitive potassium channels. tance of the gKATP channel, leading to changes in granule Annu Rev Neurosci 11:97–118, 1988 membrane potential, ion composition, or both. Verdugo 4. Babenko AP, Aguilar-Bryan L, Bryan J: A view of SUR/Kir6.X, KATP and colleagues (42,43) have reported evidence that secre- channels. Annu Rev Physiol 60:667–687, 1998 tory granules require potassium channels for appropriate 5. Seino S: ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies. Annu Rev Physiol 61:337–362, release. The gKATP channel might not only function as a 1999 channel as it does in the plasma membrane. The gKATP 6. Inagaki N, Gonoi T, Clement JPT, Namba N, Inazawa J, Gonzalez G, channel might play additional roles due to different ligand, Aguilar-Bryan L, Seino S, Bryan J: Reconstitution of IKATP: an inward

DIABETES, VOL. 52, MARCH 2003 775 INSULIN SECRETORY GRANULE KATP CHANNEL

rectiﬁer subunit plus the sulfonylurea receptor. Science 270:1166–1170, regulated secretory pathway of pancreatic ␤-cells. J Cell Sci 114:3059– 1995 3066, 2001 7. Cook DL, Satin LS, Ashford ML, Hales CN: ATP-sensitive Kϩ channels in 30. Hellman B, Sehlin J, Taljedal IB: Glibenclamide is exceptional among pancreatic ␤-cells: spare-channel hypothesis. Diabetes 37:495–498, 1988 hypoglycaemic sulphonylureas in accumulating progressively in beta-cell- 8. Clement IV JP, Kunjilwar K, Gonzalez G, Schwanstecher M, Panten U, rich pancreatic islets. Acta Endocrinol 105:385–390, 1984

Aguilar-Bryan L, Bryan J: Association and stoichiometry of KATP channel 31. The´venod F, Anderie I, Schulz I: Monoclonal antibodies against MDR1 subunits. Neuron 18:827–838, 1997 P-glycoprotein inhibit chloride conductance and label a 65-kDa protein in 9. Inagaki N, Gonoi T, Seino S: Subunit stoichiometry of the pancreatic pancreatic zymogen granule membranes. J Biol Chem 262:24410–24417, beta-cell ATP-sensitive Kϩ channel. FEBS Lett 409:232–236, 1997 1994

10. Shyng SL, Nichols CG: Octameric stoichiometry of the KATP channel 32. Sharma N, Crane A, Clement IV JP, Gonzalaz G, Babenko AP, Bryan J, complex. J Gen Physiol 110:655–664, 1997 Aguilar-Bryan L: The C terminus of SUR1 is required for trafﬁcking of KATP 11. Henquin JC: Triggering and amplifying pathways of regulation of insulin channels. J Biol Chem 274:20628–20632, 1999 secretion by glucose. Diabetes 49:1751–1760, 2000 33. Zerangue N, Schwappach B, Jan YU, Jan LY: A new ER trafﬁcking signal ϩ 12. Cook DL, Hales CN: Intracellular ATP directly blocks K channels in regulates the subunit stoichiometry of plasma membrane KATP channels. pancreatic ␤-cells. Nature 311:271–273, 1984 Neuron 22:537–548, 1999

13. Ashcroft FM, Harrison DE, Ashcroft SJ: Glucose induces closure of single 34. Schwappach B, Zerangue N, Jan YN, Jan LY: Molecular basis for KATP potassium channels in isolated rat pancreatic ␤-cells. Nature 312:446–448, assembly: transmembrane interactions mediate association of a Kϩ chan- 1984 nel with an ABC transporter. Neuron 26:155–167, 2000 14. Gembal M, Gilon P, Henquin JC: Evidence that glucose can control insulin 35. Gylfe E, Hellman B, Sehlin J, Taljedal B: Interaction of sulfonylurea with release independently from its action on ATP-sensitive Kϩ channels in the pancreatic ␤-cell. Experientia 40:1126–1134, 1984 mouse ␤ cells. J Clin Invest 89:1288–1295, 1992 36. Daniel S, Noda M, Straub SG, Sharp GW: Identification of the docked 15. Sato Y, Aizawa T, Komatsu M, Okada N, Yamada T: Dual functional role of granule pool responsible for the first phase of glucose-stimulated insulin membrane depolarization/Ca2ϩ influx in rat pancreatic ␤-cell. Diabetes secretion. Diabetes 48:1686–1690, 1999 41:438–443, 1992 37. Miki T, Nagashima K, Tashiro F, Kotake K, Yoshitomi H, Tamamoto A, 16. Gembal M, Detimary P. Gilon P, Gao ZY, Henquin JC: Mechanism by which Gonoi T, Iwanaga T, Miyazaki J-I, Seino S: Defective insulin secretion and

glucose can control insulin release independently from its action on enhanced insulin action in KATP channel-deﬁcient mice. Proc Natl Acad Sci adenosine triphosphate-sensitive Kϩ channels in mouse ␤ cells. J Clin USA95:10402–10406, 1998 Invest 91:871–880, 1993 38. Seghers V, Nakazaki M, DeMayo F, Aguilar-Bryan L, Bryan J: SUR1

17. Aizawa T, Sato Y, Ishihara F, Taguchi N, Komatsu M, Suzuki N, Hashizume knockout mice: a model for KATP channel-independent regulation of K, Yamada T: ATP-sensitive Kϩ channel-independent glucose action in rat insulin secretion. J Biol Chem 275:9270–9277, 2000 pancreatic ␤ cell. Am J Physiol 266:C622–C627, 1994 39. Komatsu M, Schermerhorn T, Noda M, Straub SG, Aizawa T, Sharp GWG:

18. Sato Y, Henquin JC: The KATP channel-independent pathway of regulation Augmentation of insulin release by glucose in the absence of extracellular of insulin secretion by glucose: in search of the underlying mechanism. Ca2ϩ: new insights into stimulus-secretion coupling. Diabetes 46:1928– Diabetes 47:1713–1721, 1998 1938, 1997 19. Detimary P, Van den Berghe G, Henquin JC: Concentration dependence 40. Henquin JC, Ishiyama N, Nenquin M, Ravier MA, Jonas JC: Signals and and time course of the effects of glucose: on adenine and guanine pools underlying biphasic insulin secretion. Diabetes 51 (Suppl. 1):S60– nucleotides in mouse pancreatic islets. J Biol Chem 271:20559–20565, 1996 S67, 2002 20. Liu M, Shapiro ME: A new method for isolation of murine islets with 41. Eliasson L, Renstro¨ m E, Ding WG, Proks P, Rorsman P: Rapid ATP- markedly improved yields. Transplant Proc 27:3208–3210, 1995 dependent priming of secretory granules precedes Ca2ϩ induced exocyto- 21. Watkins S, Geng X, Li L, Papworth G, Robbins PD, Drain P: Imaging sis in mouse pancreatic ␤ cells. J Physiol 503:399–412, 1997 secretory vesicles by fluorescent protein insertion into propetide rather 42. Nguyen T, Chin WC, Verdugo P: Role of Ca2ϩ/Kϩ ion exchange in than mature secreted peptide. Traffic 3:461–471, 2002 intracellular storage and release of Ca2ϩ. Nature 395:908–912, 1998 22. Lo¨ hrke B, Derno M, Kru¨ ger B, Viergutz T, Matthes HD, Jentsch W: 43. Quesada I, Chin WC, Steed J, Campos-Bedolla P, Verdugo P: Mouse mast Expression of sulphonylurea receptors in bovine monocytes from animals cell secretory granules can function as intracellular ionic oscillators. with a different metabolic rate. Pflu¨gers Arch-Eur J physiol 434:712–720, Biophys J 80:2133–2139, 2001 1997 44. Donelan MJ, Morfini G, Julyan R, Sommers S, Hays L, Kajio H, Briaud I, 2ϩ 23. Drain P, Li L, Wang J: KATP channel inhibition by ATP requires distinct Easom RA, Molkentin JD, Brady ST, Rhodes CJ: Ca -dependent dephos- functional domains of the cytoplasmic C terminus of the pore-forming phorylation of kinesin heavy chain on beta-granules in pancreatic beta- subunit. Proc Natl Acad SciUSA95:13953–13958, 1998 cells. Implications for regulated beta-granule transport and insulin 24. Suzuki M, Fujikura K, Kotake K, Inagaki N, Seino S, Takata K: Immuno- exocytosis. J Biol Chem 277:24232–42, 2002 localization of sulphonylurea receptor 1 in rat pancreas. Diabetologia 45. Koriyama N, Kakei M, Nakazaki M, Yaekura K, Ichinari K, Gong Q,

42:1204–1211, 1999 Morimitsu S, Yada T, Tei C: PIP2 and ATP cooperatively prevent cytosolic 25. Suzuki M, Fujikura K, Inagaki N, Seino S, Takata K: Localization of the Ca2ϩ-induced modiﬁcation of ATP-sensitive Kϩ channels in rat pancreatic ϩ ␤ ATP-sensitive K channel subunit Kir6.2 in mouse pancreas. Diabetes -cells. Diabetes 49:1830–1839, 2000 46:1440–1444, 1997 46. Baukrowitz T, Schulte U, Oliver D, Herlitze S, Tucker SJ, Ruppersbrerg JP,

26. Carpentier JL, Sawano F, Ravazzola M, Malaisse WJ: Internalization of Fakler B: PIP2 and PIP as determinants for ATP inhibition of KATP 3H-glibenclamide in pancreatic islet cells. Diabetologia 29:259–261, 1986 channels. Science 282:1141–1144, 1998

27. Ozanne SE, Guest PC, Hutton JC, Hales CN: Intracellular localization and 47. Baukrowitz T, Fakler B: KATP channels gated by intracellular nucleotides molecular heterogeneity of the sulphonylurea receptor in insulin-secreting and phospholipids. Eur J Biochem 267:5842–5848, 2000 cells. Diabetologia 38:277–282, 1995 48. Hilgemann DW, Ball R: Regulation of cardiac Naϩ,Ca2ϩ exchange and

28. Schnell Landstro¨ m AH, Westman J, Håkan Borg LA: Lysosomes and KATP potassium channels by PIP2. Science 273:956–959, 1996 pancreatic islet function: time course of insulin biosynthesis, insulin 49. Shyng SL, Nichols CG: Membrance phospholipid control of nucleotide

secretion, and lysosomal transformation after rapid changes in glucose sensitivity of KATP channel. Science 282:1138–1141, 1998 concentration. Diabetes 37:309–316, 1988 50. Simonsen A, Wurmser AE, Emr SD, Stenmark H: The role of phosphoi- 29. Molinete M, Dupuisw S, Brodsky FM, Halban PA: Role of clathrin in the nositides in membrane transport. Curr Opin Cell Biol 13:485–492, 2001

776 DIABETES, VOL. 52, MARCH 2003