Chromaffin Cell Synaptotagmin Isoforms Form Functionally And Spatially Separable Granule Pools

Chromaffin granules have long been divided into “pools” whose membership is based on functional criteria. However, what distinguishes these pools, at the molecular level, has been more difficult to define. A key difference between granules may be whether they harbor synaptotagmin-1 (Syt-1) or synaptotagmin-7 (Syt-7). We previously showed that Syt-1 and Syt-7 are rarely found together on the same granule and confer different properties to the fusion event. The major goal of this study was to assess whether Syt isoforms are sorted into functionally or spatially separable pools. A useful tool in this context is provided by monitoring the kinetics of capacitance increases in response to electrophysiological stimulation. We approximated this approach using a combination of chemical stimulation and imaging of individual fusion events. When cells were strongly and repeatedly depolarized, Syt-1 granules exhibited a greater likelihood of fusion during each stimulus cycle than Syt-7 - consistent with a difference in fusion readiness between granule populations. Granules bearing Syt isoforms were differentially dependent on Ca2+ channel activation, as revealed by blockers of channel subtypes. That spatial heterogeneity might exist with respect to where granules fuse is apparent from analyzing exocytosis in the cell footprint. Quantitative image analysis revealed that Syt-7 granules tend to fuse in closer proximity to one another than Syt-1 granules. We also analyzed the distribution of Syt-1 and Syt-7 granules within cells. Granules with Syt-1 were placed further from the cell surface and potential sites of exocytosis than those with Syt-7. Moreover, Syt-1 granules were not only more mobile than those with Syt-7, but “newcomer” granules coming from within cells accounted for a greater fraction of all Syt-1 events than for Syt-7. Overall, this study provides evidence that granule pools in chromaffin cells may be defined by selective sorting of Syt-1 or Syt-7.