Defective Regulated Exocytosis in Mast Cells from Synaptotagmin-2 Knockout Mice.

Synaptotagmins (Syts) are a family of membrane proteins present on a variety of intracellular organelles. They contain a short N terminus, a single transmembrane domain and two calcium binding C2 domains. Twelve Syt isoforms have been identified, in vertebrates. Syt-1 is the postulated calcium sensor in neuronal regulated exocytosis. Syt2 is functionally and structurally closely related to Syt1, it is the major Syt isoform in mast cells (MC) and it localizes on the MC secretory granules. MC are central effectors of allergic reactions and enhancers of host immune responses against bacterial infections. MC respond to a variety of stimuli with three effector mechanisms: secretion of preformed inflammatory mediators stored in their granules via regulated exocytosis, release of newly synthesized cytokines and chemokynes by constitutive exocytosis, and secretion of prostaglandins and leukotrienes by an exocytosis-independent mechanism. Based on the role of Syt1 in neuronal exocytosis, we postulate that Syt2 is the calcium sensor in MC regulated exocytosis. To investigate this possibility we generated a Syt2 knockout mouse using homologous recombination. Disruption of Syt2 results in low viability (around 14 days survival), growth impairment (approximately half size and weight of the control littermates) and severe ataxia and muscular weakness. We derived MC from bone marrow and lungs of 2 weeks-old Syt2^−/− mice and control littermates (Syt2^+/−, Syt2^+/+). We found no morphological or developmental difference in Syt2^−/− MC when compared with controls, and after 6 weeks in culture Syt2^−/− MC have normal surface expression of FcεRI and c-kit as measured by flow cytometry. However, we found a 50% inhibition in β-hexosaminidase and histamine release upon stimulation by IgE crosslinking, compatible with a defect in degranulation. Furthermore, other effector responses seem not to be compromised, as there was no difference in stimulated LTC4 release when compared with controls. Our data proves the essential role of Syt2 in MC regulated exocytosis. We are now performing additional functional assays and single-cell electrophysiology.