Munc18-1 and Munc18-2 Proteins Modulate β-Cell Ca2+ Sensitivity and Kinetics of Insulin Exocytosis Differently

Abstract

Fast neurotransmission and slower hormone release share the same core fusion machinery consisting of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. In evoked neurotransmission, interactions between SNAREs and the Munc18-1 protein, a member of the Sec1/Munc18 (SM) protein family, are essential for exocytosis, whereas other SM proteins are dispensable. To address if the exclusivity of Munc18-1 demonstrated in neuroexocytosis also applied to fast insulin secretion, we characterized the presence and function of Munc18-1 and its closest homologue Munc18-2 in ␤-cell stimulus-secretion coupling. We show that pancreatic ␤-cells express both Munc18-1 and Munc18-2. The two Munc18 homologues exhibit different subcellular localization, and only Munc18-1 redistributes in response to glucose stimulation. However, both Munc18-1 and Munc18-2 augment glucose-stimulated hormone release. To maintain glucose homeostasis in the body, the pancreatic ␤-cell must in a controlled way produce, store, and secrete insulin in response to appropriate stimuli (1). Regulated membrane fusion resulting in insulin exocytosis in ␤-cells is managed by the same core SNARE 3 (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) fusion machinery that mediates release of chemical signals at highly specialized central neuronal synapses (2-4). Three conserved SNARE proteins, VAMP2, syntaxin 1A, and SNAP-25, connect vesicles with the plasma membrane by forming an exceptionally stable protein complex that holds the intrinsic, but slow, capability to perform lipid bilayer fusion (5). Except for the SNARE proteins, additional cooperating proteins are critical to guarantee speed and accuracy of diverse membrane fusion events occurring in excitable cells. Such a key accessory regulator in synaptic transmission is Munc18-1, a member of the Munc18 (mammalian homologue of the unc-18 gene) family (6). In addition to Munc18-1 (with two alternatively spliced variants, Munc18-1a and Munc18-1b), Munc18-2 and Munc18-3 isoforms have also been identified (7-9). Munc18-2, also named Munc18b or muSec1 (mammalian ubiquitously Sec1), is the closest homologue of Munc18-1, showing 63% amino acid sequence identity (7).The 67-kDa hydrophilic Munc18-1 protein has been shown to be necessary not only for vesicle exocytosis but also for docking of vesicles to the plasma membrane (10). Indeed, in the absence of Munc18-1 in neuronal cells, neither evoked nor spontaneous neurotransmission can operate (6). However, besides the reported positive effect of Munc18-1 in regulated exocytosis (6, 11-13), Munc18-1 has also been identified as a negative regulator of insulin secretion (14, 15). Still, Munc18-1 is primarily considered to be a neuronal protein, whereas the homologue Munc18-2 has a wider tissue expression profile. For instance, Munc18-2 is prominent in epithelial cells (16) and has been identified as a binding partner of Cab45, a Ca 2ϩ -binding protein prominent in pancreatic acini (17). Recently Cab45/ Munc18-2 was a...

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

“…This is of importance, as first-phase GSIS is much reduced in patients with type 2 diabetes, and this has been attributed in part to greatly reduced islet SYN-1A levels [23], along with reduction of cognate SNAREs (SNAP25 and VAMP2) and the Sec1/Munc18-like protein (SM) Munc18a. SYN-3 was shown in pancreatic acinar cells to preferentially form complexes with VAMP8 [20] and Munc18b [38,39], which are also present in beta cells [40][41][42]. We recently reported, employing the Vamp8-knockout mice [43], that VAMP8 also mediated the recruitment and fusion of newcomer SGs, and was the putative cognate v-SNARE that binds SYN-3.…”

Deploying insulin granule–granule fusion to rescue deficient insulin secretion in diabetes

“…Interactions of another member of the STXBP family, STXBP3, with syntaxin 4 have been shown to influence second-phase GSIS, but the precise exocytotic event has not been elucidated [18]. A recent paper on STXBP2 reported that overexpression of this member of the STXBP family in beta cells increased calcium sensitivity to evoke a larger exocytotic response [19], implicating that this protein modulates a distinct set of SNARE proteins. Much further work will be required to elucidate the identities and functions of these as yet undefined membrane fusion SNARE molecules, the accessory proteins that modulate the assembly and disassembly of these SNARE complexes and the precise exocytotic events shown in Fig.…”

Key proteins involved in insulin vesicle exocytosis and secretion

“…ii) Certain SM proteins interact in other ways with the syntaxin protein family; the SM proteins combine with the N terminal sequence of the conserved syntaxin protein, which is termed the open state. This interaction is more common than the aforementioned interaction (6,25). iii) The direct combination of SM proteins and the SNARE complex.…”