BK channels affect glucose homeostasis and cell viability of murine pancreatic beta cells

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1 Diabetologia (211) 54: DOI 1.17/s ARTICLE BK hannels affet gluose homeostasis and ell viability of murine panreati beta ells M. Düfer & Y. Neye & K. Hörth & P. Krippeit-Drews & A. Hennige & H. Widmer & H. MClafferty & M. J. Shipston & H.-U. Häring & P. Ruth & G. Drews Reeived: 1 June 21 / Aepted: 8 September 21 / Published online: 28 Otober 21 # Springer-Verlag 21 Abstrat Aims/hypothesis Evidene is aumulating that Ca 2+ -regulated K + (K Ca ) hannels are important for beta ell funtion. We used BK hannel knokout () mie to examine the role of these K Ca hannels for gluose homeostasis, beta ell funtion and viability. Methods Gluose and insulin tolerane were tested with male wild-type and mie. BK hannels were deteted by single-ell RT-PCR, ytosoli Ca 2+ onentration ([Ca 2+ ] ) by fura-2 fluoresene, and insulin seretion by radioimmunoassay. Eletrophysiology was performed with the path-lamp tehnique. Apoptosis was deteted via aspase 3 or TUNEL assay. Results BK hannels were expressed in murine panreati beta ells. mie were normoglyaemi but displayed M. Düfer : Y. Neye : K. Hörth : P. Krippeit-Drews : P. Ruth : G. Drews () Institute of Pharmay, Department of Pharmaology and Toxiology, University of Tübingen, Auf der Morgenstelle 8, 7276 Tübingen, Germany gisela.drews@uni-tuebingen.de A. Hennige : H.-U. Häring Department of Internal Mediine, Division of Endorinology, University of Tübingen, Tübingen, Germany H. Widmer Division of Biologial and Biomedial Siene, Shool of Life Sienes, Glasgow Caledonian University, Glasgow, UK H. MClafferty : M. J. Shipston Centre for Integrative Physiology, College of Mediine and Veterinary Mediine, University of Edinburgh, Edinburgh, UK markedly impaired gluose tolerane. Geneti or pharmaologial deletion of the BK hannel redued gluose-indued insulin seretion from isolated islets. and BK hannel inhibition (with iberiotoxin, 1 nmol/l) broadened ation potentials and abolished the after-hyperpolarisation in gluose-stimulated beta ells. However, did not affet ation potential frequeny, the plateau potential at whih ation potentials start or gluose-indued elevation of [Ca 2+ ]. had no diret influene on exoytosis. Importantly, in islet ells the fration of apoptoti ells and the rate of ell death indued by oxidative stress (H 2 O 2,1 1 μmol/l) were signifiantly inreased ompared with wild-type ontrols. Similar effets were obtained with iberiotoxin. Determination of H 2 O 2 -indued K + urrents revealed that BK hannels ontribute to the hyperpolarising K + urrent ativated under onditions of oxidative stress. Conlusions/interpretation Ablation or inhibition of BK hannels impairs gluose homeostasis and insulin seretion by interfering with beta ell stimulus seretion oupling. In addition, BK hannels are part of a defene mehanism against apoptosis and oxidative stress. Keywords Apoptosis. Beta ell. BK hannel. Exoytosis. Iberiotoxin. Insulin. Stimulus seretion oupling Abbreviations BGC Blood gluose onentration BK hannel knokout C m Membrane apaitane [Ca 2+ ] Cytosoli Ca 2+ onentration K ATP hannel ATP-dependent K + hannel K Ca hannel Ca 2+ -ativated K + hannel ROS Reative oxygen speies V m Membrane potential Wild-type

2 424 Diabetologia (211) 54: Introdution Nutrient-indued insulin release ritially depends on the ativity of ion hannels and thus the extent of membrane depolarisation. The key event linking elevated gluose metabolism to alterations of eletrial ativity and eventually inreased exoytosis is the losure of ATP-dependent K + hannels (K ATP hannels) and subsequent opening of voltage-dependent L-type Ca 2+ hannels. Besides K ATP hannels, the beta ells express a variety of other K + hannels that are regulated by voltage and/or by the ytosoli Ca 2+ onentration ([Ca 2+ ] )[1 3]. The primary funtion of voltage-dependent K + hannels (K v hannels), namely the repolarisation of ation potentials, has been established for years [4]; however, the role of Ca 2+ -ativated K + hannels (K Ca hannels) is less lear. Currents through K Ca hannels of large ondutane (BK hannels) were identified eletrophysiologially in primary and lonal beta ells more than 2 years ago. Initially, these hannels were suggested to play a role in the metaboli potentiation of insulin seretion and/ or regulation of the harateristi membrane potential osillations [5, 6] but subsequent investigations did not onfirm these assumptions. Several groups have shown that membrane potential osillations are not affeted by BK hannel inhibitors [7, 8] and that the ativation of K ATP hannels, but not of K Ca hannels, is a key event for indution of the eletrially silent interburst phases [9, 1]. However, several reent observations have stimulated renewed interest in K Ca hannels as regulators of beta ell funtion: (1) studies with two mouse models laking sulfonylurea reeptor 1 (SUR1)/inward retifier K + hannel Kir 6 (Kir6.2)-omposed K ATP hannels (Sur1 [also known as Ab8]- and Kir6.2 [also known as Knj11] knokout [KO] mie, respetively) have shown that regulated insulin release is possible via a K ATP -hannel-independent pathway whih involves alterations of plasma membrane potential and [Ca 2+ ] [11, 12]; (2) the K + urrent that is ativated during eah burst phase of Ca 2+ ation potentials (K slow urrent) is not solely mediated by K ATP hannels but ontains a sulfonylureainsensitive omponent that strongly depends on [Ca 2+ ] [11, 13]; (3) K Ca hannels of small (SK1 3) and intermediate (SK4) ondutane have been suggested to ontribute to the K slow urrent [3, 14]; and (4) knokout of K Ca hannels of the SK4 type influenes in vivo gluose homeostasis [3]. Interestingly, a reent study with beta ells from human non-diabeti donors suggests that the role of BK hannels might be underestimated and provides evidene that inhibition of BK hannels affets insulin seretion [15]. The generation of BK hannel knokout () mie [16] by deletion of the Slo1 (also known as Knma1) gene now allows a detailed study of the ontribution of these K + hannels to beta ell physiology and regulation of gluose homeostasis. Our data show that BK hannels are involved in ation potential repolarisation. We demonstrate, for the first time, that loss of BK hannels impairs insulin seretion and glyaemi ontrol. In addition, inreases the sensitivity of beta ells to oxidative stress via a diret effet on ell viability. Methods Animals, ell and islet preparation Experiments were performed with and wild-type () mie (in-house breeding). The priniples of laboratory animal are were followed (National Institutes of Health publiation number 85-23, revised 1985). Experiments were arried out aording to German laws (Regierungspräsidium Stuttgart, approval number M 8/3). mie were generated as previously desribed [16]. Mie were killed by CO 2 and islets were isolated by ollagenase digestion, dispersed in Ca 2+ -free medium and ultured for up to 4 days (RPMI 164 medium, 11.1 mmol/l gluose, supplemented with 1% [vol./vol.] fetal alf serum, 1 U/ml peniillin and 1 μg/ml streptomyin). Solutions and hemials Bath solution for [Ca 2+ ],membrane potential (V m ) and apaitane (C m ) measurements omprised: 14 mmol/l NaCl, 5 mmol/l KCl, 1.2 mmol/l MgCl 2, 2.5 mmol/l CaCl 2, 15 mmol/l gluose and 1 mmol/l HEPES, ph 7.4. Pipette solution for perforated-path reordings omprised: 1 mmol/l KCl, 1 mmol/l NaCl, 7 mmol/ lk 2 SO 4,4mmol/lMgCl 2, 2 mmol/l CaCl 2, 1 mmol/l EGTA, 5 mmol/l HEPES, ph 7.15, and amphoteriin B, 25 μg/ml. Pipette solution for inside-out reordings omprised: 13 mmol/l KCl, 1.2 mmol/l MgCl 2, 2 mmol/l CaCl 2, 1 mmol/l EGTA, 2 mmol/l HEPES, ph 7.4. Bath solution omprised: 13 mmol/l KCl, 1 mmol/l EDTA, 2 mmol/ l HEPES, ph 7.2, with free Ca 2+ adjusted to 1 μmol/l by CaCl 2. Pipette solution for C m determination omprised: 135 mmol/l K-gluonate, 1 mmol/l EGTA, 4 mmol/l MgCl 2, 5 mmol/l HEPES, 3 mmol/l Na 2 ATP,.2 mmol/l AMP, ph 7.2, with free Ca 2+ adjusted to 1 μmol/l. Inubation medium for insulin seretion: 122 mmol/l NaCl, 4.8 mmol/l KCl, 2.5 mmol/l CaCl 2, 1.1 mmol/l MgCl 2, 1 mmol/l HEPES and.5%(wt/vol.) bovine serum albumin, ph 7.4. Fura-2-aetoxymethyl ester (fura-2am) was from Moleular Probes (Eugene, OR, USA) and iberiotoxin was from Bahem (Bubendorf, Switzerland). RPMI 164 medium was from PromoCell (Heidelberg, Germany) and peniillin/streptomyin was from GIBCO/BRL (Karlsruhe, Germany). All other hemials were purhased from Sigma (Deisenhofen, Germany) and Merk (Darmstadt, Germany). Gluose and insulin tolerane tests In vivo experiments were performed with male mie and littermates aged weeks. Gluose (2 g/kg body weight) or

3 Diabetologia (211) 54: insulin (1 U/kg body weight) was injeted intraperitoneally. Plasma gluose onentration was monitored for 12 (gluose tolerane) or 6 (insulin sensitivity) min. Mie were fasted for 16 h before testing gluose tolerane. Measurement of intraellular free [Ca 2+ ] [Ca 2+ ] was measured by the fura-2 method using equipment and software from TILL photonis (Gräfelfing, Germany). Cells were identified as beta ells when [Ca 2+ ] was not dereased by 15 mmol/l gluose as desribed for alpha ells [17]. Cells were loaded with fura-2am (5 μmol/l) for 3 min at 37 C. [Ca 2+ ] was alulated following an in vitro alibration with fura-2 K + -salt [18]. Eletrophysiology Path pipettes were pulled from borosiliate glass apillaries (Clark, Pangbourne, UK). V m was reorded at 32 C (EPC-9 path-lamp amplifier; HEKA, Lambreht, Germany). Cells were identified as beta ells when they were eletrially silent with.5 mmol/l gluose but showed Ca 2+ ation potentials after swithing to 15 mmol/l gluose. K + urrents were eliited by 1 mv voltage steps (3 ms) from a holding potential of 7 mv. C m was determined in the standard whole-ell onfiguration. To assay exoytosis an 83 Hz sine wave with a peak-to-peak amplitude of 3 mv was applied to the ells ( 7 mv holding potential). A train of eight yles was applied to the ell every 5 s. C m,membrane ondutane and aess ondutane were derived from analysis of the sinusoidal membrane urrent at two orthogonal phase angles by the LokIn extension of the Pulse software ( sine+d protool, HEKA). Data were analysed with Chart software (ADInstruments, Spehbah, Germany). Insulin seretion Bathes of five islets were inubated for 6 min at 37 C. Insulin was determined by radioimmunoassay using rat insulin (Lino Researh, St Charles, MI, USA) as the standard. Single ell PCR Cellular ontents of single ells were aspirated into RNAse-free borosiliate path-pipettes ontaining 7 μl of RNAse-free water and were immediately transferred to an Eppendorf tube for DNA synthesis using Sensisript reverse transriptase (Qiagen, Crawley, UK), RNasin ribonulease inhibitor (Promega, Southampton, UK) and a mix of random and poly-dt primers in a final volume of 2 μl at 37 C for 1 h. For PCR analysis, 2 5 μl of single-ell DNA was used in a 2 μl reation using GoTaq DNA polymerase (Promega). Primers, insulin: forwards 5 -CAGCAAGCAGGTCATTGTTT-3, reverse 5 -CAGTAGTTCTCCAGCTGGTAGA-3. Primers for the BK hannel alpha subunit spanned the site of spliing C2 to detet BK hannel splie variants: forwards 5 - GTTTGTGAGCTGTGTTTTGTG-3, reverse5 -CTACGGT TACCAGGTGGTCATGT-3. Amplions were run on a 1.5% (wt/vol.) agarose gel and visualised using Sybr Safe. Determination of apoptoti islet ells Islet ells were seeded on glass over slips and ultured in RPMI 164 medium for 24 h. Apoptosis was determined by ative aspase 3 (NuView assay, Biotium, Hayward, CA, USA) or TUNEL staining. In eah ondition, a minimum of 1, ells from three to four different isolations was ounted. Growth medium was removed and 4 μl DEVD-NuView 488 aspase 3 substrate was added. Upon enzymati leavage of the substrate, the released DNA dye migrates to the ell nuleus where it binds to the DNA resulting in a highly fluoresent omplex. For TUNEL labelling, panreati islet ells were fixed with 3% (wt/vol.) paraformaldehyde at 2 25 C for 1 h. After rinsing with PBS, beta ells were permeabilised for 2 min on ie [.1% [wt/vol.] Triton- X and sodium itrate solution). Eah sample was overed with 5 μl TUNEL reation mixture and inubated in a humidified atmosphere for 1 h at 37 C in the dark. TUNEL-positive ells were deteted by fluoresein staining (48 nm) and the number of total ells was visualised with Hoehst Presentation of results At least three different ell preparations were used for eah series. Means ± SEM are given in the text. Statistial signifiane of differenes was assessed by a one-sample or Student's t test for paired values; multiple omparisons were made by ANOVA followed by Student Newman Keuls test. For ation potential harateristis five ation potentials of eah experiment were averaged. Peak values were set to t= ms and data were analysed every 5 ms within the preeding and following 2 ms. A p value of less than.5 was onsidered signifiant. Results Ativity and expression of BK hannels in panreati islet ells In exised inside-out pathes of isolated islet ells unitary K + urrent amplitudes of 11.±.8 pa (holding potential of 5 mv, symmetrial K + onentration), with an open probability (Po) of.12±.2 (n=5), were deteted in 16 out of 43 pathes (Fig. 1a). The urrent was Ca 2+ dependent and the ord ondutane alulated from the single hannel I/V urve was 238±8 ps (n=5, Fig. 1). As a Ca 2+ -regulated K + urrent with similar properties was absent in exised pathes of beta ells obtained from BK- KO mie (n=3, Fig. 1b) the single hannel urrents were attributed to BK hannels. The expression of BK hannel pore-forming alpha subunits in panreati islet ells was further haraterised by single-ell PCR experiments

4 426 Diabetologia (211) 54: a 1 µmol/l Ca 2+ Ca 2+ -free 1 b 1 µmol/l Ca 2+ 2 pa 3 min I (pa) V m (mv) 2 3 d bp 8 Cell 1 No RT Cell 2 NTC 6 4 BK 4 Insulin 2 Fig. 1 BK hannel ativity and expression in panreati islet ells. a Single hannel urrents were reorded at a holding potential of j5 mv in inside-out pathes of islet ells. For the time indiated by the horizontal bar, the BK-hannel-positive pathes were perifused with Ca 2+ -free solution. One representative experiment out of 16 single hannel reordings is shown. represents the losed state, 1 represents the open state of one BK hannel. b Single hannel urrents were reorded at a holding potential of j5 mv in inside-out pathes of islet ells. One representative experiment out of 3 single hannel reordings is shown. I/V urve for single hannel urrents reorded from inside-out pathes of beta ells at voltages ranging from 8 to +3 mv. d mrna of the BK hannel splie variants Zero (~6 bp) and Strex (~8 bp) were deteted by RT-PCR performed with ytosol derived from single islet ells. Cell 1 and 2 are examples of insulin-positive ells. NTC, non-template ontrol; No RT, ontrol without enzyme (Fig. 1d). mrnas of two BK hannel alpha subunit splie variants (Zero and Strex) were deteted in insulin-positive islet ells, indiating BK hannel expression in beta ells. mie display redued insulin seretion and impaired gluose tolerane To eluidate whether BK hannels are involved in regulation of insulin seretion, the seretory response to gluose was determined in stati inubations of isolated and islets (Fig. 2a). Insulin release was similar in and KO islets under resting onditions (3 mmol/l gluose) and at gluose onentrations lower than 1 mmol/l. However, stimulation

5 Diabetologia (211) 54: a Insulin seretion (% of ontent) G 6G 8G 1G 15G 3G b Plasma gluose (mmol/l) Time (min) Plasma gluose (% of t ) Time (min) Fig. 2 Effet of on insulin seretion, gluose tolerane and insulin sensitivity. a Isolated islets were inubated with different gluose onentrations for 6 min. In the presene of 1, 15 and 3 mmol/l gluose, insulin release from islets was signifiantly lower than from islets (n=4 different preparations per genotype). b BGC of male and littermates was monitored before and for 2 h after i.p. injetion of gluose (2 g/kg body weight). For determination of insulin sensitivity, blood gluose was followed before and for 1 h after i.p. injetion of insulin (1 U/kg body weight). Gluose tolerane of mie was markedly impaired ompared with mie (n=6 and n=8 C57Bl6 littermates, respetively), whereas insulin sensitivity was unaffeted (n=6 and n=5 littermates, respetively). p.5, p.1; blak bars and solid lines, mie; white bars and dashed lines, mie with 1, 15 and 3 mmol/l gluose was muh less effetive in islets ompared with ontrols (p.5, n=4 separate preparations per genotype). and islets showed no differene in insulin seretion indued by 3 mmol/l K + in the presene of 25 μmol/l diazoxide (.9±.2 ng ml 1 islet 1 ;.9±.5 ng ml 1 islet 1, n=3). The differenes in gluose-mediated insulin release were not due to alterations in insulin ontent ( 25±1 ng/islet; 27±3 ng/islet, n=4 per genotype). The inhibitory effet of was mimiked in islets treated with the BK hannel blokers iberiotoxin (1 nmol/l) or paxillin (1 μmol/l). Insulin seretion of islets stimulated with 15 mmol/l gluose was redued from 1.9±.2 to.8±.2 and.7±.1 ng ml 1 islet 1, respetively, when iberiotoxin or paxillin were present during the 6 min inubation period (n=3, p.5). To test whether the impaired funtion of islets affets glyaemi ontrol we monitored blood gluose onentration (BGC) in response to gluose and insulin hallenge, respetively (Fig. 2b, ). BGC was similar in and mie fed ad libitum or after overnight fasting (fed 1.3±.3 mmol/l for vs 1.4±.5 mmol/l for mie; fasted 7.3±.5 mmol/l for and 7.8±.6 mmol/l for mie; n=8 and n=6, respetively). However, an intraperitoneal gluose tolerane test revealed signifiant differenes between the two genotypes. In response to the gluose hallenge (2 g/kg body weight) BGC of mie was markedly higher ompared with mie 15, 3 and 6 min after injetion. Insulin (1U/kg body weight) injeted i.p. redued the BGC to the same extent in and mie, respetively. These data demonstrate that the redution of insulin seretion aused by BK hannel ablation leads to impaired gluose homeostasis in vivo. Stimulus seretion oupling in beta ells The observation that gluose-stimulated insulin release was redued in islets whereas K + -indued seretion was unaffeted suggests that loss of BK hannel funtion may impair the oupling of gluose metabolism, eletrial ativity and Ca 2+ influx. Consequently, we tested whether the stimulus seretion asade was altered in BK hanneldefiient beta ells. The key event linking gluose metabolism to exoytosis is the inrease in [Ca 2+ ] indued by opening of L-type Ca 2+ hannels subsequent to membrane depolarisation. Therefore, we investigated whether ablation of BK hannels affeted [Ca 2+ ]. In beta ells the inrease in gluose onentration from.5 to 15 mmol/l led to an initial drop of [Ca 2+ ] due to ativation of ATP-dependent Ca 2+ pumps. With the opening of L-type Ca 2+ hannels [Ca 2+ ] was elevated to a plateau and, finally, harateristi osillations ourred (n=12). This pattern of ativity likewise existed in beta ells (n=14, Fig. 3). Therewasnodiffereneintheareaundertheurveforthe first rise of [Ca 2+ ] after elevating gluose from.5 to 15 mmol/l (Fig. 3d f) or in the frequeny of Ca 2+ osillations (Fig. 3g). The lag time between the elevation of gluose onentration and the rise of [Ca 2+ ] ( 14± 15 s [n=12] vs 146±13 s [n=13]) was also similar for both genotypes. Compatible with the data obtained for [Ca 2+ ] neither the resting membrane potential in.5 mmol/l gluose nor the plateau potential (potential from whih Ca 2+ ation potentials start) with 15 mmol/l gluose were signifiantly altered by (Fig. 4a,b). On average, the resting membrane potential was 69±1 mv (n=1) in beta ells and 7±1 mv (n=12) in beta ells. The plateau potential was 47±1 mv (n=12) and 48±1 mv (n=12), respetively. Interestingly, influened the shape of Ca 2+

6 428 Diabetologia (211) 54: a b [Ca 2+ ] (nmol/l) [Ca 2+ ] (nmol/l) [Ca 2+ ] (nmol/l) d AUC Ca (µmol/l min) e [Ca 2+ ] (nmol/l) 3 15G f 15G 3 [Ca 2+ ] (nmol/l) 2 min 2 min g Ca 2+ osillations (min 1 ) Fig. 3 Influene of on [Ca 2+ ]. a does not affet basal [Ca 2+ ] in.5 mmol/l gluose (n=14 for both genotypes). b d After swithing to 15 mmol/l gluose there was no differene in the first Ca 2+ peak in vs ells in terms of: (b) the initial derease (n=14 for both genotypes); () the peak [Ca 2+ ] ( n=12; n=15); or (d) the area under the urve ( n=12; n=13). e, f Representative experiments for (e) ells, and (f) BK- KO ells. g A summary of the analysis of the frequeny of Ca 2+ osillations in the presene of 15 mmol/l gluose ( n=51; n=23) ation potentials. In beta ells, the spike duration of ation potentials at half-maximum amplitude was signifiantly inreased from 12±1 ms (, n=12) to 18±1 ms (, n =12, p.1) and the typial afterhyperpolarisation was ompletely abolished (Fig. 4). Idential hanges ould be indued in ells by addition of 1 nmol/l of the speifi BK hannel bloker iberiotoxin (n=4, not shown), indiating that BK hannels are involved in ation potential repolarisation. However, did not affet ation potential frequeny ( 58±1 min 1 [n=12], 62±16 min 1 [n=1]) or ation potential amplitude ( 6±2 mv; 63±3 mv [n=12]). Furthermore, we examined whether BK hannels diretly interat with the exoytoti mahinery. The standard wholeell onfiguration was used to measure C m and ells were dialysed with pipette solution adjusted to 1 μmol/l free Ca 2+.Figure4d shows that the rate of hange in C m in response to 1 μmol/l Ca 2+ was not signifiantly different between (n=14) and (n=13) beta ells. To exlude that has any effet on ell size, whole-ell apaitane was determined in eah experiment ( 9.7±.5 pf [n=13]; 9.6±.5 pf [n=14]) and exoytosis was normalised to these values. BK hannels affet beta ell viability As BK hannels have been desribed to be involved in regulation of ell survival [19] we determined apoptoti ell death in and ells ultured in 11.1 mmol/l gluose. Importantly, the fration of apoptoti islet ells was more than doubled in vs mie (Fig. 4e). In agreement with the inreased rate of ell death in islets, treatment of ells with iberiotoxin (1 nmol/l, 36 h) elevated apoptosis by ~5% (n=3, p.5, not shown). This suggests that, in addition to the modulation of eletrial ativity, BK hannels are involved in pathways determining apoptoti ell death. Role of BK hannels in response to oxidative stress As BK hannel ativity is linked to ell death we studied the role of these hannels in oxidant-indued K + hannel ativation and apoptosis. In previous studies we demonstrated that stimulus seretion oupling of panreati beta ells is severely affeted by oxidative stress [2 22]. Reative oxygen speies (ROS) suh as H 2 O 2 hyperpolarise the plasma membrane via ativation of K ATP hannels, thereby inhibiting insulin seretion. As we ould also show that abrogation of eletrial ativity oinides with a drasti inrease in [Ca 2+ ] [2], ativation of K Ca hannels might also ontribute to the hyperpolarising urrent in addition to K ATP hannels. To test this hypothesis we measured wholeell K + urrents (Fig. 5a,b). In beta ells stimulated with 15 mmol/l gluose, appliation of 1 mmol/l H 2 O 2 evoked a marked inrease in K + urrent that was largely inhibitable by tolbutamide (1 μmol/l, n=8, p.1). However, a small omponent resistant to the K ATP hannel bloker was also identified. This omponent was sensitive to paxillin (1 μmol/l, n=4, p.1), a bloker of BK hannels [23]. By ontrast, in beta ells the urrent

7 Diabetologia (211) 54: a V m (mv) b V m (mv) G 15G.5G 15G 1 min 1 min V m (mv) Time (s) d Δ C m (ff s 1 pf 1 ) e Apoptoti islet ells (% of total ells) Fig. 4 Eletrial ativity, exoytosis and ell viability. a,b The effet of gluose stimulation on eletrial ativity is similar in (a) and (b) BK- KO beta ells. Gluose onentrations are indiated by the horizontal bars, with the white setion indiating gluose.5 mmol/l and the hathed setion indiating gluose 15 mmol/l. Experiments were performed in the perforated-path onfiguration. The reordings are representative of 12 experiments with eah genotype. Comparison of gluose-indued ation potentials reorded from (dotted line) and (solid line) beta ells. The traes show the average of 12 experiments per genotype. d Exoytosis was determined as the inrease in C m immediately after establishing the standard whole-ell onfiguration ( n=14; n=13). The ells were dialysed with pipette solution ontaining 1 μmol/l Ca 2+. e mie have an inreased number of apoptoti islet ells. Apoptoti ells were deteted by determination of ativated aspase 3. Isolated islet ells were analysed after overnight ulture in RPMI medium (11.1 mmol/l gluose). The data were obtained from four different preparations per genotype. p.5 evoked by 1 mmol/l H 2 O 2 was signifiantly smaller and ompletely abolished by 1 μmol/l tolbutamide (n=4, p.1). Data are summarised in Fig. 5. These results demonstrate that urrent through BK hannels ontributes to the H 2 O 2 -indued hyperpolarisation of panreati beta ells. As, for other tissues, ativation of BK urrent has been shown to be an important mehanism to redue ROSindued ell damage [24], we tested whether geneti deletion or pharmaologial inhibition of BK hannels affets the rate of apoptosis in response to H 2 O 2. Figure 5d shows that the inrease in the fration of apoptoti islet ells provoked by 1, 25 and 1 μmol/l H 2 O 2, respetively, was signifiantly lower in ompared with BK- KO ells (n=3), suggesting that ativation of BK hannels is part of the ellular defene mehanism to maintain ell viability under onditions of elevated oxidative stress. To test whether BK hannel blokade with iberiotoxin ould mimi the effet of, islet ells were inubated for 36 h with 1 nmol/l iberiotoxin prior to H 2 O 2 appliation. In this series of experiments the pro-apoptoti ation of 1 μmol/l H 2 O 2 was markedly enhaned in iberiotoxin-treated ells vs ontrols (n=3, Fig. 5e). Disussion BK hannels are Ca 2+ - and voltage-regulated K + hannels that our in most tissues of the body. In exitable ells of endorine, nervous and vasular systems BK hannels link intraellular signalling to eletrial ativity [16, 25 27]. In 1996 DNA of the Slo1 gene that enodes the poreforming alpha subunit of BK hannels was identified and haraterised in human panreati islets [28]. In the present study we deteted two splie variants of the alpha subunit, Zero and Strex, in single beta ells (Fig. 1d). In human beta ells, BK urrent has been reported to aount for a signifiant part of K v urrents and pharmaologial inhibition of BK hannels influened by insulin seretion [15]. However, the signifiane of these observations for glyaemi ontrol of the whole organism remains unlear. The

8 43 Diabetologia (211) 54: a b 15G, 1 mmol/l H 2 O 2 15G, 1 mmol/l H 2 O 2 I (pa)75 1 µmol/l tolbutamide 1 µmol/l paxillin 6 min 1 min I (pa)75 1 µmol/l tolbutamide 1 min I (pa) Tolbutamide Paxillin d Apoptoti islet ells (% of total ells) Apoptoti islet ells (% of total ells) H 2 O 2 (µmol/l) H 2 O 2 (µmol/l) e Fig. 5 Influene of H 2 O 2 on ion urrents and ell viability in and islet ells. a, b The K + urrent indued by appliation of H 2 O 2 was omposed of K ATP and BK urrent. In the perforated-path onfiguration gluose-stimulated beta ells (15 mmol/l gluose) were exposed to 1 mmol/l H 2 O 2 (indiated by the white horizontal bar). In ells from islets the urrent evoked by 1 mv depolarising voltage steps from the holding potential of 7 mv was inhibited by tolbutamide (1 μmol/l; indiated by the blak horizontal bar) exept for a small omponent that was sensitive to the BK hannel inhibitor paxillin (1 μmol/l; indiated by the hathed horizontal bar). b In ells from islets the urrent was ompletely bloked by tolbutamide. The diagram shows the analysis of the experiments desribed in a, b; blak bars, ; white bars,. The number of experiments for H 2 O 2 -indued inrease in K + urrent was 13 for and seven for beta ells. The effet of tolbutamide was tested in eight and four ells, respetively. Paxillin was given in addition to the sulfonylurea in four experiments with ells. d The fration of aspase-3-positive islet ells after 1 h inubation with different onentrations of H 2 O 2 (1, 25 and 1 μmol/l, respetively) was signifiantly higher in BK hannel-defiient islet ells. The experiments were performed after overnight ulture of dispersed islet ells in RPMI medium (G11.1). The diagram summarises the data obtained from three separate preparations per genotype. Blak bars, ; white bars,. e Islets of mie were inubated in RPMI medium or in medium supplemented with 1 nmol/l iberiotoxin for 36 h. For an additional 6 h H 2 O 2 (25 or 1 μmol/l) was added and the fration of apoptoti ells was determined by TUNEL staining (n= 3). Blak bars, ; white bars, + iberiotoxin. p.5, p.1, p.1 vs or vs iberiotoxin-treated ells generation of mie enabled us to investigate the impat of this hannel on regulation of BGC and insulin release. We demonstrate for the first time that loss of BK hannels affets gluose homeostasis in vivo. did not alter BGC of fasted mie or of animals fed ad libitum but markedly impaired gluose tolerane in response to an intraperitoneal gluose hallenge. This effet ould be asribed to a redution of gluose-stimulated insulin release (Fig. 2). The fat that mie displayed redued insulin seretion without any hange in insulin ontent pointed to an impairment of beta ell funtion. Evaluation of gluoseevoked eletrial ativity revealed that did not affet plateau potential or ation potential frequeny but broadened single ation potentials and abolished the afterhyperpolarisation (Fig. 4). Importantly, we obtained similar effets by pharmaologial inhibition of BK hannels in beta ells. As the path-lamp experiments were performed with single ells or small lusters that do not display the harateristi osillations reorded from whole islets, the eletrophysiologial data annot rule out that BK- KO affets gluose-indued burst frequeny. However, this is very unlikely as the frequeny of Ca 2+ osillations, whih is ontrolled by V m, is similar in both genotypes. Our experiments show that inhibition or KO of BK hannels does not influene bulk [Ca 2+ ] (Fig. 3). However, BK hannels might partiipate in regulation of the loal Ca 2+ onentration whih is deisive for ontrol of the exoytoti mahinery [29]. The existene of sub-membrane Ca 2+ gradients with high Ca 2+ onentration diretly beneath the plasma membrane has been shown for primary beta ells by Quesada et al. [3]. In addition, the loss of the after-hyperpolarisation might leave more Ca 2+ hannels in the inativated state thereby reduing the number of hannels that ould be reruited by the following ation potential. Suh subtle hanges in Ca 2+ influx are most likely too small to hange bulk [Ca 2+ ], but ould alter exoytosis of insulin-ontaining granules by diminishing [Ca 2+ ] in sub-membrane domains. BK hannels are known to partiipate in the ontrol of ell mass. For various tumour ell lines it has been shown that BK hannel ativity modulates proliferation and ell death [19, 31]. Our data provide the first evidene that BK hannel ablation affets ell viability in panreati islets

9 Diabetologia (211) 54: (Fig. 4e). Compared with islet ells, the fration of apoptoti ells was more than doubled in ells. Importantly, similar hanges ourred in ells after inubation with iberiotoxin. This indiates that alterations in ell viability are not a by-produt of the geneti manipulation but are diretly linked to loss of BK hannel funtion. The mehanisms by whih BK hannels modulate signalling pathways determining ell death are not yet resolved. Mitohondrial BK hannels have been reported to interfere with Ca 2+ sequestration [32] and the mitohondrial permeability transition pore [33, 34]. However, the fat that the non-membrane-permeant peptide iberiotoxin indues similar effets as argues against an involvement of mitohondrial BK hannels. It is well known that, in several ell types, plasma-membrane-loated K + hannels ontribute to the regulation of apoptosis. Mostly, inhibition of K + hannels redues apoptosis but the opposite has also been reported [35, 36]. There are several studies demonstrating that pharmaologial or geneti elimination of K ATP hannels inreases apoptoti ell death [22, 37]. Our results suggest that the same holds true for onditions with redued BK hannel ativity. As neither insulin ontent nor high K + -indued insulin seretion was diminished in islets, the proapoptoti effet of BK hannel elimination is unlikely to ontribute to the impaired seretory response indued by gluose stimulation. However, our study demonstrates that BK hannels are important regulators of beta ell viability under onditions of inreased oxidative stress (Fig. 5). It is well known that beta ells are extremely vulnerable to ROS due to their poor antioxidant defene mehanisms [38]. Consequently, oxidative stress severely impairs beta ell funtion and viability [39 42]. BK hannels have been reported to be modulated by H 2 O 2 [43]. Our data show that besides K ATP hannels [44], ativation of BK hannels ontributes to the hyperpolarising urrent eliited in the presene of H 2 O 2 (Fig. 5a, b) whih might serve as a protetive mehanism to avoid Ca 2+ overload of the ells. At present we annot rule out that knokout of BK hannels indues hanges seondary to hannel deletion. As the paxillin-sensitive omponent of the H 2 O 2 -indued K + urrent is muh smaller than the differene between and beta ells, expression of additional K + urrents might be affeted. So far suh interations have not been desribed for panreati beta ells but are reported for the ohlea, where leads to disappearane of Kv7.4 hannels in outer hair ells [45]. Importantly, ompared with ontrols the suseptibility to H 2 O 2 -mediated apoptosis was markedly elevated in islet ells derived from mie or in iberiotoxin-treated ells (Fig. 5d,e). This suggests that ativation of BK hannels ontributes to the defene mehanisms proteting beta ells against oxidative ell damage. In agreement with our results it was demonstrated for hippoampal neurons that pharmaologial BK hannel inhibition aggravates hypoxia-indued ell death [24]. It is noteworthy that inhibition of BK or K ATP hannels inreases the rate of basal apoptosis but exerts ontrary effets on the sensitivity of beta ells to oxidative stress. The protetive effet of K ATP hannel inhibition is aused by an upregulation of antioxidant enzymes that depends on alterations in intraellular Ca 2+ sequestration [22]. As limitation of BK hannel ativity does not oinide with protetion against ROS-indued ell death, this indiates that modulation of antioxidative defene mehanisms is partiularly related to K ATP hannels and not generally indued by K + hannel inhibition. In summary, our investigations show that BK hannels play a role in gluose homeostasis and affet the suseptibility of panreati beta ells to oxidative stress. Aknowledgements We thank I. Breuning for skilful tehnial assistane. This work was supported by grants from the DFG Dr225/ 6-3 (G. Drews), DU425/1-2 (M. Düfer) and the Wellome Trust (M. J. Shipston and P. Ruth). Parts of this study have been published in abstrat form (Diabetologia [24] 47(Suppl 1):28). Duality of interest The authors delare that there is no duality of interest assoiated with this manusript. Referenes 1. MaDonald PE, Wheeler MB (23) Voltage-dependent K + hannels in panreati beta ells: role, regulation and potential as therapeuti targets. Diabetologia 46: Tamarina NA, Wang Y, Mariotto L et al (23) Small-ondutane alium-ativated K + hannels are expressed in panreati islets and regulate gluose responses. Diabetes 52: Düfer M, Gier B, Wolpers D, Krippeit-Drews P, Ruth P, Drews G (29) Enhaned gluose tolerane by SK4 hannel inhibition in panreati beta-ells. 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