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 2

Local calcium influx at exocytosing granules in INS1 cells.

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Local calcium influx at exocytosing granules in INS1 cells. (A) Cumulati...
(A) Cumulative exocytosis events (orange) and cell-averaged lyn-R-GECO-Ca2+ fluorescence (Fcell/Fcell0, black) in an INS1 cell periodically stimulated with K+, as indicated. See Supplemental Figure 2 for cell images. (B) Frequency of exocytosis events in 15 cells as in A, relative to the most recent K+ pulse. (C) Average images of lyn-R-GECO fluorescence centered on granules undergoing exocytosis or not (Failure) and temporally aligned to the onset of application of 75 mM K+ (pink arrowhead); 68 granules each in 15 cells. (D) As in C, but for cells stimulated with 250 μM ACh (24 granules each in 10 cells). (E) As in C, but for cells expressing the II–III loop fragment and stimulated with 75 mM K+ (30 granules each in 9 cells). Arrowheads indicate onset of stimulation. Scale bar: 2 μm (C–E). (F) Average lyn-R-GECO-Ca2+ fluorescence at granules (F/F0) undergoing exocytosis (responders, blue), failures (red), and random locations (black) during the first K+ pulse. The cells were loaded with EGTA-AM, and 75 mM K+ was applied as indicated (12 cells with 67 responders and 200 failures). (G) As in F, but for cells preloaded with BAPTA-AM (14 cells, 43 granules each). (H) As in F, but for cells expressing the II–III loop fragment and stimulated for 2 seconds (9 cells, 30 granules each). For comparison, the signal at responders in untransfected cells is shown in gray (8 cells, 45 granules). (I) As in F, but for cells stimulated with ACh for 2 seconds (10 cells, 24 granules each).