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Priming of insulin granules for exocytosis by granular Cl^- uptake and acidification

¹ Department of Physiological Sciences, Lund University, Sölvegatan 19, SE-223 62 Lund, Sweden
² University of Chicago, Department of Neurobiology, Pharmacology and Physiology, 947 E. 58^th Street, MC 0926, Chicago, IL 60637, USA
³ Physiologisches Institut, Universität des Saarlandes, D-66421 Homburg/Saar, Germany

View larger version (14K): [in a new window]   Fig. 1. Intracellular Cl- fluxes control Ca2+-elicited exocytosis. (A) Increases in cell capacitance (C) evoked by infusion of the Ca2+- and ATP-containing control solution (ctrl, n=34) and in presence of niflumic acid (nifl, n=12) or DIDS (n=7). (B) Average rates of capacitance increase (C/t) ± s.e.m. (C) C evoked as in (A) at 165 mM (n=10), 40 mM (n=8), and 2 mM (n=9) intracellular [Cl-] as indicated. (D) Average C/t ± s.e.m. Statistical significance was evaluated using Student’s t-test. *P<0.05; ***P<0.001.

View larger version (36K): [in a new window]   Fig. 2. Antibody characterization and ClC-3 expression in mouse B-cells. Immunoblots of mouse islet homogenate and cells overexpressing ClC-3 isoforms. (A) Whole-cell lysates (10 µg/lane), crude membrane and cytosol isolates (100 µg/lane) were prepared from tsA cells stably transfected with full-length hClC-3 or vector alone (mock), or from mouse islets. Anti-hClC-3c detected a 120 kDa protein in the membrane fractions of the transfected cells. After deglycosylation with N-glycanase for 18 hours immunoreactivity was detected as a single band at 90 kDa in the tsA cells. In islets, both the glycosylated form (double band at 115-130 kDa) as well as the deglycosylated protein (90 kDa) were detected. (B) Isoform specificity of anti-hClC-3c was verified by immunoblotting of deglycosylated whole-cell lysates (10 µg/lane) obtained from HEK 293 cells stably expressing hClC-3, hClC-1, hClC-4 or rat ClC-2. (C) and (D), as in (A) and (B), except that anti-hClC-3n was used.

View larger version (85K): [in a new window]   Fig. 3. ClC-3 channels localize to the B-cell granule membrane. (A) Distribution of anti-hClC-3c binding, insulin immunoreactivity and overlay of anti-hClC-3c and insulin immunoreactivity visualized by confocal immunocytochemistry. Bar, 2 µm. (B) Immunogold detection of ClC-3 localization to insulin-containing secretory granules (SG) visualized by electron microscopy. The 12 nm gold particles appear as small black dots (arrow). Bar, 100 nm. Ly, lysosome.

View larger version (25K): [in a new window]   Fig. 4. Exocytosis requires granular acidic pH. (A) Capacitance increases (C) elicited by infusion of the Ca2+- and ATP-containing control electrode solution (ctrl, n=21), after addition of sucrose (sucr, n=6) and in the presence of CCCP (n=6) or nigericin (nig, n=5). (B) Average rates of capacitance increase (C/t) ± s.e.m. (C) C elicited by the control solution (ctrl, n=9), in the presence of the Na+/H+-exchanger inhibitor EIPA (n=10) and the v-H+-ATPase inhibitor bafilomycin (bafi, n=11). (D) Mean C/t ± s.e.m. ***P<0.001 **P<0.01.

View larger version (6K): [in a new window]   Fig. 5. Tolbutamide accelerates intragranular acidification. Granule acidification evoked by tolbutamide estimated as the relative increase (in per cent) in the initial LysoSensor-fluorescence signal (F/F0) after addition of the compound.

View larger version (24K): [in a new window]   Fig. 6. KATP-channel effectors modulate exocytosis by influencing granular pH. (A) Increases in cell capacitance (C) under control conditions (red, n=26), after addition of Mg-ADP (green, n=12), tolbutamide and Mg-ADP (blue, n=12), diazoxide (cyan, n=19) or DIDS (magenta, n=8). (B) Average rate of changes in cell capacitance (C/t) ± s.e.m. **P<0.01; ***P<0.001. (C) Changes in granular pH estimated as the relative changes (in per cent) of the initial LysoSensor-fluorescence intensity (F/F0) after establishment of the standard whole-cell configuration, using the Ca2+- and ATP-containing control solution (red, n=5), after addition of ADP (green, n=5), tolbutamide and ADP (blue, n=5) and DIDS (magenta, n=5) The arrow indicates the establishment of the standard whole-cell configuration. (D) Average changes (per cent of initial) in Lysosensor fluorescence after 60 seconds recording (F/F0). Data are mean values ± s.e.m. ***P<0.001. Statistical significances were evaluated comparing the responses in the respective groups with the responses obtained with the ATP-containing control (red). (E), as in (C), but after supplementing the control pipette solution with: CCCP (red, n=14), ADP and CCCP (green, n=9), tolbutamide, ADP and CCCP (blue, n=10), diazoxide and CCCP (cyan, n=7), DIDS and CCCP (magenta, n=5), anti-hClC-3c and CCCP (yellow, n=9) and anti-hClC-3n and CCCP (brown, n=6). The arrow indicates the establishment of the standard whole-cell configuration. (F), as in (D), but statistical significance was evaluated by comparing the responses in the respective groups with the responses obtained when CCCP was added to the control solution (red). dz, diazoxide; tb, tolbutamide. Data are mean values ± s.e.m. **P<0.01, ***P<0.001.

View larger version (15K): [in a new window]   Fig. 7. Granular Cl- fluxes and pH affect the recruitment of secretory granules for release. (A-B) Increases in cell capacitance (C) evoked by a train of ten voltage-clamp depolarizations from -70 mV to zero (500 milliseconds, 1 Hz) under control conditions (n=9), after addition of Mg-ADP (n=4), in the presence of simultaneously added tolbutamide (tb) and Mg-ADP (n=6), after inclusion of anti-hClC-3c (n=7), anti-hClC-3n (n=17) or CCCP (n=6) as indicated. (B) Average total increases in cell capacitance (C) ± s.e.m. for experiments in (A). *P<0.05; **P<0.01, ***P<0.001.

View larger version (29K): [in a new window]   Fig. 8. Model for the molecular mechanisms underlying regulated granular Cl- fluxes and their actions on intragranular pH and exocytosis. (A) Replenishment of the RRP of secretory granules depends on intragranular acidification. (B) Metabolically regulated granular Cl- uptake through an ion channel complex comprising of ClC-3 Cl- channels (grey) and a mdr1-like 65 kDa regulatory protein (white), determines the rate of the intragranular pH decrease driven by the V-type H+-ATPase (black). Tolbutamide (tb), diazoxide (dz) and ADP are likely to exert their actions by interacting with the regulatory 65 kDa protein, whereas ATP, DIDS and anti-hClC-3c bind to ClC-3 directly. See text for details.

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