CAPS-1 requires its C2, PH, MHD1 and DCV domains for dense core vesicle exocytosis in mammalian CNS neurons

Linda Van Keimpema,Matthijs Verhage,Ruud F Toonen,Robbelien Kooistra

doi:10.1038/s41598-017-10936-4

Linda Van Keimpema, Matthijs Verhage + Show 2 more

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https://doi.org/10.1038/s41598-017-10936-4

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Journal: Scientific Reports	Publication Date: Sep 7, 2017
Citations: 21	License type: open-access

Affiliation: Sylics (Netherlands)

Abstract

CAPS (calcium-dependent activator protein for secretion) are multi-domain proteins involved in regulated exocytosis of synaptic vesicles (SVs) and dense core vesicles (DCVs). Here, we assessed the contribution of different CAPS-1 domains to its subcellular localization and DCV exocytosis by expressing CAPS-1 mutations in four functional domains in CAPS-1/-2 null mutant (CAPS DKO) mouse hippocampal neurons, which are severely impaired in DCV exocytosis. CAPS DKO neurons showed normal development and no defects in DCV biogenesis and their subcellular distribution. Truncation of the CAPS-1 C-terminus (CAPS Δ654-1355) impaired CAPS-1 synaptic enrichment. Mutations in the C2 (K428E or G476E) or pleckstrin homology (PH; R558D/K560E/K561E) domain did not. However, all mutants rescued DCV exocytosis in CAPS DKO neurons to only 20% of wild type CAPS-1 exocytosis capacity. To assess the relative importance of CAPS for both secretory pathways, we compared effect sizes of CAPS-1/-2 deficiency on SV and DCV exocytosis. Using the same (intense) stimulation, DCV exocytosis was impaired relatively strong (96% inhibition) compared to SV exocytosis (39%). Together, these data show that the CAPS-1 C-terminus regulates synaptic enrichment of CAPS-1. All CAPS-1 functional domains are required, and the C2 and PH domain together are not sufficient, for DCV exocytosis in mammalian CNS neurons.

Highlights

Calcium-dependent activator protein for secretion (CAPS) is an important regulator of dense core vesicle (DCV) exocytosis
To test if CAPS DKO neurons show developmental defects, which could influence the outcome of our functional assays, we analyzed neuronal development in hippocampal CAPS DKO neurons compared to CAPS-2 KO control neurons
In this study we investigated which CAPS-1 domains are important for the function of CAPS-1 in mammalian dense core vesicle exocytosis

Summary

Introduction

Calcium-dependent activator protein for secretion (CAPS) is an important regulator of dense core vesicle (DCV) exocytosis. Deletion of CAPS-1 and CAPS-2 expression (CAPS DKO) severely reduces DCV exocytosis in mammalian neurons and exocytosis of secretory granules in chromaffin cells[8,9,10,11]. The C-terminal part of CAPS-1 appears to be important for CAPS-1 interaction with DCVs in PC12 cells[16] Mutations in these domains interfere with CAPS-1 function in calcium-dependent DCV exocytosis in PC12 cells[6, 16, 21,22,23,24] and DCV exocytosis in C. elegans[25]. A natural CAPS-2 splice isoform, which lacks the MHD1 and DCV domains, rescues exocytosis in CAPS DKO chromaffin cells[10]. CAPS DKO neurons showed a drastic decrease in DCV release probability, which was rescued by expression of wild type CAPS-1 but not CAPS-1 C2 or PH domain mutants or the C-terminal truncation. Our study shows that all CAPS-1 functional domains are essential for DCV exocytosis in mammalian CNS neurons

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