Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes
Nikhil R. Gandasi, Peng Yin, Michela Riz, Margarita V. Chibalina, Giuliana Cortese, Per-Eric Lund, Victor Matveev, Patrik Rorsman, Arthur Sherman, Morten G. Pedersen, Sebastian Barg
Nikhil R. Gandasi, Peng Yin, Michela Riz, Margarita V. Chibalina, Giuliana Cortese, Per-Eric Lund, Victor Matveev, Patrik Rorsman, Arthur Sherman, Morten G. Pedersen, Sebastian Barg
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Research Article Cell biology Endocrinology

Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes

Abstract

Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II–III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

Authors

Nikhil R. Gandasi, Peng Yin, Michela Riz, Margarita V. Chibalina, Giuliana Cortese, Per-Eric Lund, Victor Matveev, Patrik Rorsman, Arthur Sherman, Morten G. Pedersen, Sebastian Barg

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Figure 4

L-type Ca2+ channels and Munc13 cluster at docked insulin granules.

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L-type Ca2+ channels and Munc13 cluster at docked insulin granules. (A a...
(A and B) Images showing parts of INS1 or human β cells coexpressing EGFP-CaV1.2 or Munc13-EGFP as indicated (A), together with the granule marker NPY-mCherry (B). Solid circles indicate granules with associated CaV1.2/Munc13 clusters, and dotted circles indicate granules without the cluster. Conditions for INS1 (21–52 cells) cells are: control (3 mM glucose); IRES vector control [bicistronic p(empty)IRES-NPY24 mCherry]; overexpression of the Munc13 C2-domain fragment using the IRES vector (M13); the CaV1.2 II–III loop fragment (II–III loop) using the IRES vector; and long-term exposure to 0.5 mM oleate or palmitate. Conditions for human β cells are: 3 or 10 mM glucose (3G, 10G) in ND (20–34 cells, 3 donors) or T2D (31–52 cells, 3 donors) cells. Scale bars: 1 μm. (C) Average images of EGFP-CaV1.2 or Munc13-EGFP spatially aligned to the location of docked granules; conditions as in A and B. The number of analyzed granules is shown in yellow. Scale bar: 1 μm. (D–F) Quantification of EGFP-CaV1.2 or Munc13-EGFP clusters shown in A and B as (D) the percentage of granules associated with a cluster, (E) granule-associated fluorescence (ΔF/S), and (F) cluster density. The ΔF/S for EGFP-CaV1.2 was essentially zero at random locations (–0.004 ± 0.001, 38 cells; P < 0.0001, by Student’s t test). *P < 0.05 and ***P < 0.001, by Student’s t test. (G) Example of a granule docking in INS1 cells and corresponding Munc13-EGFP (M13) or EGFP-CaV1.2 signals (separate cells). Scale bar: 1 μm. (H) Quantification of granule (gray) and corresponding Munc13-EGFP (blue) or EGFP-CaV1.2 signals (green) aligned to the moment of docking (34 and 21 granules in 12 and 9 cells, respectively).