Distinct roles of the C₂A and the C₂B domain of the vesicular Ca²⁺ sensor synaptotagmin 9 in endocrine β-cells

Synaptotagmins form a family of calcium-sensor proteins implicated in exocytosis, and these vesicular transmembrane proteins are endowed with two cytosolic calcium-binding C₂ domains, C₂A and C₂B. Whereas the isoforms syt1 and syt2 have been studied in detail, less is known about syt9, the calcium sensor involved in endocrine secretion such as insulin release from large dense core vesicles in pancreatic β-cells. Using cell-based assays to closely mimic physiological conditions, we observed SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-independent translocation of syt9C₂AB to the plasma membrane at calcium levels corresponding to endocrine exocytosis, followed by internalization to endosomes. The use of point mutants and truncations revealed that initial translocation required only the C₂A domain, whereas the C₂B domain ensured partial pre-binding of syt9C₂AB to the membrane and post-stimulatory localization to endosomes. In contrast with the known properties of neuronal and neuroendocrine syt1 or syt2, the C₂B domain of syt9 did not undergo calcium-dependent membrane binding despite a high degree of structural homology as observed through molecular modelling. The present study demonstrates distinct intracellular properties of syt9 with different roles for each C₂ domain in endocrine cells.