ABSTRACT
Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.
Subject(s)
Exocytosis/physiology , Glucagon-Secreting Cells/metabolism , Glucagon/metabolism , Intracellular Calcium-Sensing Proteins/physiology , Synaptotagmins/physiology , Action Potentials/physiology , Animals , Blood Glucose/drug effects , Calcium Channels/metabolism , Exocytosis/drug effects , Gene Expression/genetics , Glucagon/blood , Glucagon/genetics , Glucagon/pharmacology , Glucagon-Secreting Cells/ultrastructure , Hypoglycemia/blood , Insulin/pharmacology , Islets of Langerhans/metabolism , Male , Mice , Mice, Inbred Strains , Mice, Knockout , omega-Conotoxins/pharmacologyABSTRACT
Synchronous neurotransmission depends on the tight coupling between Ca(2+) influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.
