The Ca2+ Affinity of Synaptotagmin 1 Is Markedly Increased by a Specific Interaction of Its C2B Domain with Phosphatidylinositol 4,5-Bisphosphate

Anand Radhakrishnan,Alexander Stein,Reinhard Jahn,Dirk Fasshauer

doi:10.1074/jbc.m109.042499

Abstract

The synaptic vesicle protein synaptotagmin 1 is thought to convey the calcium signal onto the core secretory machinery. Its cytosolic portion mainly consists of two C2 domains, which upon calcium binding are enabled to bind to acidic lipid bilayers. Despite major advances in recent years, it is still debated how synaptotagmin controls the process of neurotransmitter release. In particular, there is disagreement with respect to its calcium binding properties and lipid preferences. To investigate how the presence of membranes influences the calcium affinity of synaptotagmin, we have now measured these properties under equilibrium conditions using isothermal titration calorimetry and fluorescence resonance energy transfer. Our data demonstrate that the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin. PI(4,5)P2 binding is confined to the C2B domain but is not affected significantly by mutations of a lysine-rich patch. Together, our findings lend support to the view that synaptotagmin functions by binding in a trans configuration whereby the C2A domain binds to the synaptic vesicle and the C2B binds to the PI(4,5)P2-enriched plasma membrane.

Highlights

C2 domains are Ca2ϩ binding modules of ϳ130 amino acids, first described as the second conserved region of protein kinase
The relatively low intrinsic Ca2ϩ affinities of both C2 domains are difficult to reconcile with the role of synaptotagmin 1 as the Ca2ϩ sensor for fast and synchronous neurotransmitter release, suggesting that interaction with phospholipids contributes to its Ca2ϩ sensitivity
This penetration was corroborated by electro-paramagnetic resonance (EPR) spectroscopy studies, which showed that the penetration depth increased when both C2 domains of synaptotagmin 1 were attached to each other [21] as compared with the single domains [22, 23]

Summary

Introduction

C2 domains are Ca2ϩ binding modules of ϳ130 amino acids, first described as the second conserved region of protein kinase. These data confirm that the introduced point mutations completely abolish Ca2ϩ binding to the mutated C2 domains of synaptotagmin and no auxiliary calcium binding sites are present in this protein.

Results

Conclusion