Characterization of phogrin-GCaMP6m-XC, a label to measure perigranular Ca2+-concentrations

Background and aims The beta cell and the other endocrine cells of the pancreatic islet release their respective hormones by triggered exocytosis. The final fusion step results from the influx of Ca2+ via voltage-dependent Ca2+-channels. However, metabolic stimuli like glucose release more insulin than purely depolarizing stimuli even though the Ca2+ increase is often less marked. To get a clearer view on the exocytosis-relevant Ca2+-concentration a site-specific label is needed.

Methods The recently published genetically engineered Ca2+ indicator (GECI) GCaMP6m-XC was fused to the transmembrane granule protein phogrin, so that the GECI should be localized at the outside of the insulin granule. Insulin-secreting MIN6 cells were transiently transfected with the granule-bound GECI or with the normal, cytosolic GECI and the fluorescence was measured by live cell imaging.

Results While the intensity of the cytosolic GECI was circa fivefold brighter than the one of the granule-bound GECI, the kinetic response to depolarisation by 40 mM KCl was closely similar and the relative increase in fluorescence was even higher with the granule-bound GECI. When MIN6-cells transfected with granule-bound GECI and C-peptide-mCherry were depolarized by 15 mM KCl, the increase of the GECI was markedly lower than the one elicited by 40 mM KCl, whereas the fluorescence of the cargo-directed label remained unchanged. The latter may therefore be used to indicate membrane vicinity in TIRF microscopy.

Conclusion Phogrin-GCaMP6m-XC reports perigranular Ca2+-concentration with a wide dynamic range and may be used to report the fusion-relevant submembrane Ca2+-concentration by TIRF microscopy.

Publication History

Article published online:
26 May 2022