Rapid-response unit

In an emergency situation, a speedy response is essential — whether it's at the level of an ambulance responding to an emergency phone call or a reflex response of the nervous system to a painful stimulus. Nerves communicate with one another by the Ca²⁺-induced exocytosis of synaptic vesicles and, in Nature Structural and Molecular Biology, Chapman and colleagues now provide insights into how this event can occur so rapidly.

Synaptotagmin I is anchored to synaptic-vesicle membranes by its transmembrane domain, and it can sense Ca²⁺ levels through its two cytoplasmic C2 domains (C2A and C2B). This Ca²⁺ sensing is thought to be important for the Ca²⁺-induced fusion of docked vesicles. A putative effector of synaptotagmin-I function is phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂), which has recently been shown to be selectively localized to the plasma membrane. Chapman and co-workers therefore studied the interaction of synaptotagmin I with PtdIns(4,5)P₂-containing membranes.

The authors first assessed the ability of the fragments C2A, C2B and C2A–C2B to interact with liposomes containing varying amounts of PtdIns(4,5)P₂. In the presence of Ca²⁺, C2A only interacted with liposomes containing high levels of PtdIns(4,5)P₂, which might not be physiologically relevant, whereas C2A–C2B and C2B interacted with liposomes containing low levels of PtdIns(4,5)P₂. Notably, the latter two fragments also interacted with these liposomes in the absence of Ca²⁺, which indicates that C2B might bind to PtdIns(4,5)P₂ prior to the Ca²⁺ signal in vivo.

In response to Ca²⁺, the Ca²⁺-binding loops of the C2A and C2B domains have been shown to penetrate membrane bilayers, and Chapman and colleagues inserted fluorescent probes into the four loops of C2A–C2B to see if this is the case for PtdIns(4,5)P₂-containing membranes. They used liposomes containing PtdIns(4,5)P₂ and membrane-embedded, fluorescence-quenching labels to show that, in response to Ca²⁺, three of the four loops of C2A–C2B penetrate the bilayer. By contrast, in the absence of Ca²⁺, C2A–C2B binding occurs in the absence of quenching, which indicates that Ca²⁺-independent binding does not require membrane penetration. In fact, the authors showed that this binding occurs through a polybasic region on the side of C2B.

So, does this 'pre-binding' increase the speed of the Ca²⁺-induced response of synaptotagmin I? When the authors premixed C2A–C2B with PtdIns(4,5)P₂-containing liposomes in the absence of Ca²⁺, the response of C2A–C2B to Ca²⁺ occurred with submillisecond kinetics and was more rapid than when the premixing step was omitted. Furthermore, they showed that PtdIns(4,5)P₂ steers synaptotagmin I towards PtdIns(4,5)P₂-containing membranes. Synaptotagmin I reconstituted into proteoliposomes interacts preferentially with PtdIns(4,5)P₂-containing membranes in trans; this interaction occurs in the absence of Ca²⁺ and is enhanced by Ca²⁺.

Chapman and co-workers therefore propose a model in which the side of the C2B domain first steers synaptotagmin I to PtdIns(4,5)P₂-containing membranes by interacting with PtdIns(4,5)P₂ in a Ca²⁺-independent manner. As a result of this pre-binding, in response to Ca²⁺, the C2 domains only need to be reorientated, so Ca²⁺-binding loops can penetrate the bilayer rapidly. Synaptotagmin-I–PtdIns(4,5)P₂ interactions are therefore required to steer the membrane-penetration activity of synaptotagmin I towards the plasma membrane, as well as to increase the speed of the Ca²⁺-induced response of synaptotagmin I.