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Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes
Nikhil R. Gandasi, … , Morten G. Pedersen, Sebastian Barg
Nikhil R. Gandasi, … , Morten G. Pedersen, Sebastian Barg
Published May 8, 2017
Citation Information: J Clin Invest. 2017;127(6):2353-2364. https://doi.org/10.1172/JCI88491.
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Research Article Cell biology Endocrinology

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

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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 5

Single-molecule analysis of CaV1.2 behavior.

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Single-molecule analysis of CaV1.2 behavior.
(A) Single-molecule imaging...
(A) Single-molecule imaging of EGFP-CaV1.2 at 50 Hz. Part of an INS1 cell expressing EGFP-CaV1.2 at low levels to facilitate observation of single molecules, at 2 different time points (1–2, bandpass filtered for clarity). Granules and trajectories of individual EGFP-CaV1.2 molecules with granule positions overlaid (large circles). Scale bar: 1 μm. (B) Histogram of single molecule distance traveled per frame (50 ms). The red line is a fitted diffusion equation with D1 = 0.007 and D2 = 0.035 μm2/s as diffusion coefficients; blue lines show the 2 components of the fit. The green line is the best fit, assuming a single diffusion coefficient. (C) Cumulative histograms of single-molecule residence times within circles of 100 nm diameter and centered at either granule (black) or random positions (gray). (D) Superresolution image obtained by plotting the area density of detected single molecules from a live cell. The granule positions are shown as circles. Scale bar: 0.5 μm.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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