Beschreibung:
<jats:p> In chromaffin cells, the kinetics of fusion pore expansion vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives release. Our recent studies have shown that fusion pores of granules harboring Syt-1 expand more rapidly than those harboring Syt-7. Here we sought to define the structural specificity of synaptotagmin action at the fusion pore by manipulating the Ca<jats:sup>2+</jats:sup>-binding C2B module. We generated a chimeric Syt-1 in which its C2B Ca<jats:sup>2+</jats:sup>-binding loops had been exchanged for those of Syt-7. Fusion pores of granules harboring a Syt-1 C2B chimera with all three Ca<jats:sup>2+</jats:sup>-binding loops of Syt-7 (Syt-1:7C2B<jats:sub>123</jats:sub>) exhibited slower rates of fusion pore expansion and neuropeptide cargo release relative to WT Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites than WT protein. We speculate that the Syt-1:7 C2B<jats:sub>123</jats:sub> and WT Syt-1 are likely to differ in their interactions with Ca<jats:sup>2+</jats:sup> and membranes. Subsequent in vitro and in silico data demonstrated that the chimera exhibits a higher affinity for phospholipids than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma membrane, and the rate at which it releases the membrane, contribute in important ways to the rate of fusion pore expansion. </jats:p>