Fig 1: Effect of Ano1 inhibitors on the membrane potential of β-cell from whole mice islet stimulated with 16.7 mM glucose. a, b, g Representative zero-current nystatin-perforated patch-clamp voltage recordings. Sampling rate, 18 kHz; 2-kHz filter setting. Dotted lines represent zero-voltage level. a Glucose-stimulated cell (16.7 mM glucose). b Glucose-stimulated cell ± 100 μM T-AO1 in the bathing medium. c Effect of T-AO1 (n = 3) on action potential (AP) rate, in presence of glucose. d Effect of T-AO1 (n = 3) on AP peak, in presence of glucose. e Effect of T-AO1 (n = 3) on AP amplitude, in presence of glucose. f Effect of T-AO1 (n = 3) on the membrane plateau potential during active phase in presence of glucose. g Glucose-stimulated cell ± 100 μM TA in the bathing medium. h Effect of TA (n = 7) on action potential rate, in presence of glucose. i Effect of TA (n = 9) on AP peak, in presence of glucose. j Effect of TA (n = 9) on AP amplitude, in presence of glucose. k Effect of TA (n = 9) on the membrane plateau potential during active phase, in presence of glucose. The experiments were performed on ten preparations of mice islets. Friedman test on f, P = 0.05; repeated measures ANOVA test on k, P = 6.88 × 10−10. *P < 0.05, ***P < 0.001 vs. 2.8-mM glucose condition; °°P < 0.01, °°°P < 0.001 vs. 16.7-mM glucose condition (paired Student’s t tests in c–e, h–j; Wilcoxon type tests with Dunn–Bonferroni correction in f; least significant difference tests in k)
Fig 2: Insulin release from rat pancreatic islets is drastically reduced in the presence of Ano1 inhibitors and blocking Ano1 antibodies. Islets were first preincubated in 2.8 mM glucose, in the presence (or not) of T-AO1, TA, or blocking Ano1 antibodies (ab72984), and then incubated for 90 min in the same medium with different glucose concentrations (2.8, 8.3, and 16.7 mM). The experiments were always performed on groups of eight islets isolated from preparations of two rats. a Effect of T-AO1 and TA (100 μM) on glucose-stimulated insulin secretion (GSIS) from rat islets in Hepes-buffered NaCl solution without bicarbonate as used in the patch-clamp voltage measurements (n = 58–59 from six preparations in conditions without inhibitors, n = 29–30 from three preparations in conditions with inhibitors). Preincubation with T-AO1 or TA completely abolished the increment in insulin output on glucose 8.3-mM stimulation. With 16.7 mM glucose, no increase was observed in the presence of T-AO1, while the relative increase was weak with TA (32.6 ± 10.6 %). b Dose-dependent effect of T-AO1 on 16.7 mM GSIS from rat islets in Hepes and bicarbonate-buffered NaCl solution (n = 19–20 from three preparations except for 10 μM T-AO1, n = 32). T-AO1 100 μM prevented any increase in GSIS. c Effect of rabbit blocking Ano1 antibodies ab72984 (in whole serum) on 16.7 mM GSIS from rat islets in Hepes and bicarbonate-buffered NaCl solution. c1 No antibody/no serum (n = 20 from two preparations). c2 ab72984 or serum 1:250 and c3 ab72984 or serum 1:100 (c2 and c3, n = 9–12 from three preparations). The relative increment in presence of 1:100 antibodies was reduced to 23.9 ± 16.0 % with a secretion value not significantly different from control (P = 0.15 vs. 2.8 mM glucose). One-way ANOVA test on a, P = 1.41 × 10−46; Kruskal–Wallis test on b, c2, c3, P < 0.001; Mann–Whitney test on c1, P < 0.001. *** P < 0.001 vs. 2.8 mM glucose condition; °P < 0.05, °°°P < 0.001 vs. 16.7 mM glucose-stimulated condition (Sidak tests in a; Mann–Whitney-type tests with Dunn–Bonferroni correction in b, c2, c3)
Fig 3: Effect of Ano1 inhibitors on the membrane potential of 16.7 mM glucose-stimulated murine dispersed β-cells. Zero-current nystatin-perforated patch-clamp voltage recordings performed on dispersed β-cells stimulated with 16.7 mM glucose before incubation with T-AO1 or TA (100 μM) in the bathing medium. Sampling rate, 18 kHz; 2-kHz filter setting. Dotted lines represent zero-voltage level. a–f Experiments carried out on rat dispersed β-cells, n = 7 from three preparations of rat dispersed islet cells in presence of T-AO1 and n = 8 from two preparations of rat dispersed islet cells with TA. a Representative recording ± T-AO1. Effect of T-AO1 b on AP rate and c on average membrane potential. d Representative recording ± TA. Effect of TA e on AP rate and f on average membrane potential. g–i Experiments carried out on mice dispersed β-cells, n = 4 from one preparation of mice dispersed islet cells. g Representative recording ± T-AO1. Effect of T-AO1 h on AP rate and i on average membrane potential. Repeated measures ANOVA test on c, P = 0.00123; f, P = 2 × 10−4; Friedman test on i, P = 0.18. ***P < 0.001 vs. 2.8 mM glucose condition; °°P < 0.01, °°°P < 0.001 vs. 16.7 mM glucose condition (least significant difference tests in c, f; paired Student’s t tests in b, e, h)
Fig 4: Chloride currents from rat β-cells (inside-out excised macropatches and whole cell). a–i Chloride currents from inside-out single excised patches. Pipette and bath solutions contained 150 mM NMDG-Cl. Sampling rate, 25 kHz; 2-kHz filter setting. Filled pipette resistance, 5 MΩ. Current traces recorded were induced by 1500-ms voltage steps from −100 to +100 mV, spaced 20 mV (holding potential, −70 mV, 200 ms before and after each step). Dotted lines indicate zero-current level. a–c Representative recordings of Cl− currents. The cytosolic face was exposed to bath solutions with different [Ca2+]: 0 μM in a, 1 μM in b, and 2 μM in c. d Steady-state current–voltage relationships of Cl− currents at 0 μM Ca2+ (n = 12), 1 μM Ca2+ (n = 5), and 2 μM Ca2+ (n = 8). e Representative recording of Cl− currents stimulated by 2 μM Ca2+ in the presence of T-AO1 10 μM in the pipette. f Steady-state current–voltage relationships of Cl− currents at 2 μM Ca2+ in the absence (n = 8) or presence (n = 12) of T-AO1 10 μM in the pipette and at 0 μM Ca2+ (n = 12). g, h Representative recordings of Cl− currents stimulated by 2 μM Ca2+, respectively, in the presence of boiled or active blocking Ano1 antibodies ab72984 1:100 in the pipette. i Steady-state current–voltage relationships of Cl− currents at 2 μM Ca2+ in the presence of boiled (n = 18) or active (n = 13) blocking Ano1 antibodies ab72984 1:100 in the pipette and at 0 μM Ca2+ (n = 12). j–t Whole-cell Cl− currents from dispersed β-cells. Pipette and bath solutions contained 150 mM NMDG-Cl except in anion selectivity experiments where the bath Cl− was replaced by NO3 − or Br− for repeated measures. Sampling rate, 10 or 25 kHz; 2-kHz filter setting. Filled pipette resistance, 5 MΩ. Dotted lines indicate zero current or Px/PCl = 1 level. j, l Representative current traces from β-cells induced by voltage ramps (20 mV/s) at 1 μM Ca2+ (pipette). Bath NMDG-Cl solution was replaced by either NMDG-NO3 in j or NMDG-Br in l. k Nitrate and bromide anions shift the reversal potential (V rev) toward negative values (n = 11 and 9, respectively). m Permeability ratios (Px/PCl) of nitrate and bromide anions calculated from the shifts of the reversal potentials in k using Goldman, Hodgkin, and Katz equation. n–t Current traces recorded were induced by 400-ms voltage steps from −100 to +100 mV, spaced 20 mV (holding potential, −70 mV, 100 ms before and after each step). n–p Representative recordings of whole-cell Cl− currents at 0 μM Ca2+ in n, 1 μM in o, and 1 μM in the presence of T-AO1 10 μM in the bath medium in p. q Normalized current–voltage relationships of whole-cell Cl− currents at 1 μM Ca2+ in the absence (n = 21) or presence (n = 15) of T-AO1 10 μM in the bath medium and at 0 μM Ca2+ (n = 14). r, s Representative recordings of whole-cell Cl− currents evoked by 1 μM Ca2+ at +100 mV after 6-, 8-, and 10-min membrane rupture in the presence of boiled Ano1 antibodies ab72984 1:100 in the pipette (r) or active Ano1 antibodies ab72984 1:100 in the pipette (s). t Normalized whole-cell Cl− currents evoked by 1 μM Ca2+ at the end of the +100-mV voltage step in the presence of boiled Ano1 antibodies ab72984 1:100 or active Ano1 antibodies ab72984 1:100 in the pipette after 6 min (n = 14 and 13), 8 min (n = 12 and 12), 10 min (n = 10 and 10), and 12 min (n = 5 and 8) membrane rupture. Experiments of Fig. 5 were carried out on six preparations of rat dispersed islet cells. Kruskal–Wallis tests on f, i, q, P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control (Mann–Whitney-type tests with Dunn–Bonferroni correction in f, i, q, paired Student’s t tests in k, independent Student’s t tests in t)
Fig 5: Detection of Ano1 in pancreas and pancreatic islets. a RT-PCR of cDNA prepared from mRNA extracted from rat and human tissues. Transcripts of the expected size for Ano1 are observed (rat: 223 bp; human 314 bp). The 300-bp band is shown in the molecular weight marker column (MWM). Positive control: kidney. Negative control (Neg. control): no DNA. The sequencing of PCR products confirmed 100 % identity with the reference sequence for rat Ano1 cDNA complementary of rat Ano1 mRNA. b Western blot of Ano1 in rat islets, from left to right: molecular weight column (MW) showing the 100-, 150-, and 250-kDa bands, 80 μg rat islet lysate, 30 μg rat islet lysate, and 30 μg human thyroid lysate (positive control). Ano1 is detected at 119 kDa. c Immunofluorescence staining of pancreas section. c1 Immunohistochemical labeling (green-fluorescent Tyramide Alexa 488) of Ano1 in a section photomicrograph of rat pancreas. Most of the islet cells and acinar cells (at the level of apical pole) are labeled. c2 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c1. c3 Specificity control: immunohistochemical labeling of Ano1 in a section photomicrograph of rat pancreas. The primary goat Ano1 antibodies (sc-69343) were coincubated in the presence of Ano1 synthetic peptide (ab97423) in a ratio 1:8. The labeling disappears. c4 Counterstaining labeling by hematoxylin–eosin performed on the slice used for c3. Arrows show islets. Scale bar is 50 μm
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