Electrostatic regulation of the cis- and trans-membrane interactions of synaptotagmin-1

Abstract

Synaptotagmin-1 is a vesicular protein and Ca2+ sensor for Ca2+-dependent exocytosis. Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis-interaction, thus preventing the trans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor the cis- and trans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10–100 μM. High PIP2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2 concentrations control the Ca2+-cooperativity of synaptotagmin-1. Our data provide evidence that Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1.

Requirement of Cholesterol for Calcium‐Dependent Vesicle Fusion by Strengthening Synaptotagmin‐1‐Induced Membrane Bending

“…[ 43a ] iii) Synaptotagmin‐1 binding to the planar membranes generates higher membrane bending energy that drives vesicle fusion. [ 43a ] Simulations support our previous data that PIP 2 is essential for triggering synaptotagmin‐1‐mediated Ca 2+ ‐dependent vesicle fusion by inducing the trans ‐interaction of synaptotagmin‐1 through the polybasic patch [ 25a,b,27 ] (Figure 7a,b).…”

“…The increases in PIP 2 concentration in PM‐liposomes shifted Ca 2+ titration curves to the left, indicating an increase in the Ca 2+ ‐sensitivity of C2AB membrane binding (Figure 4g and Figure S3, Supporting Information); EC 50 : 68.1 µ m Ca 2+ for 1% PIP 2 and 14.3 µ m Ca 2+ for 5% PIP 2 . High PIP 2 concentration increases Ca 2+ sensitivity for vesicle fusion; [ 25a,27 ] this interaction implies that an increase in the magnitude of the negative electrostatic potential in the plasma membranes increases the attraction of Ca 2+ ‐bound synaptotagmin‐1. Even with 5% PIP 2 in PM‐liposomes, Ca 2+ still failed to increase LDCV fusion without cholesterol (Figure 4h).…”

“…Indeed, the C2A and C2B domains bind to V‐liposomes (0% PIP 2 , 15% PS included), but this interaction was completely disrupted at physiological ionic strength due to the electrostatic effect of Mg 2+ and ATP (Figure 4i). [ 25a,b,27 ] Both the C2A and C2B domains bind to the plasma membrane to drive vesicle fusion in a physiological ionic environment, [ 25a,b,27 ] proposing that membrane binding of both the C2A and C2B domains have a cooperative and synergistic effect on membrane deformation. Taken together, C2AB binding into the plasma membrane directly provides the driving force to lower the energy barrier for Ca 2+ ‐triggered vesicle fusion to occur.…”

“…Synaptotagmin‐1 mediates Ca 2+ ‐dependent vesicle fusion by i) membrane insertion and ii) interacting with PIP 2 through the polybasic patch, which induces the trans ‐interaction of synaptotagmin‐1. [ 25a,b,27 ] Cholesterol strengthens the plasma membrane deformation and bending caused by synaptotagmin‐1 insertion. Blue in synaptotagmin‐1, the polybasic patch interacting with PIP 2 ; magenta, Ca 2+ .…”

“…[ 38 ] For example, smaller vesicles,SUVs (Figure S4e,f, Supporting Information) and SVs (Figure 5f,g), have greater bending energy per unit of surface area and accordingly show robust basal fusion efficiency compared to large vesicles. The C2AB domain of synaptotagmin‐1 is inserted into the plasma membrane [ 27,28 ] and the membrane binding energy of the C2AB domain is ≈18 k B T [ 39 ] ; the binding leads to local membrane bending and deformation. [ 29 ] This curved plasma membrane has high bending energy, which can be released to drive fusion with vesicle membranes [ 29,38 ] (Figure 7b,c).…”