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 3

Modeling of Ca2+ influx.

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Modeling of Ca2+ influx. (A) Modeled GECO/Ca2+ signal, assuming 15 L-typ...
(A) Modeled GECO/Ca2+ signal, assuming 15 L-type channels in the center and either endogenous buffering or added EGTA (1 mM). Arrowheads indicate the onset of stimulation. Image frames are shown for every 0.1 second. Scale bar: 2 μm. (B) Modeled time course of the [Ca2+] (black) and GECO signal (green) in a circle with a diameter of 75 nm and centered on a cluster of 15 Ca2+ channels, assuming no added EGTA. The [Ca2+] average over 0.1 second time intervals is shown in gray. (C) Theoretical GECO rise times (color coded) as a function of the Ca2+ channel number in the cluster and the distance from the cluster’s center. (D) Cumulative histograms of GECO rise times for responders (blue) and failures (red) for the experiments depicted in Figure 2G (P = 0.00012, by Wilcoxon Mann-Whitney U test). (E) Exocytosis probability, normalized to the probability at d = 0.1 μm, as a function of the distance to the Ca2+ channel cluster; based on data in Figure 2, C and F, and Supplemental Figure 3 and time-to-event statistics and assuming no added buffering (solid line) or 1 mM EGTA (dotted line).