Figure 4.

The R398Q,R399Q mutation impairs synaptotagmin-1/membrane interactions. (a) The R398Q,R399Q mutation does not impair the ability of the C₂AB fragment to displace a complexin-1 fragment from membrane-anchored SNARE complexes. Supported phospholipid bilayers containing reconstituted SNARE complexes were deposited into microchannels, a rat complexin-1 fragment (residues 26-83) labeled with BODIPY-FL was bound to the SNARE complexes, and displacement of the labeled complexin 1 fragment by increasing concentrations of C₂AB fragment (black circles) or C₂AB-R398Q,R399Q fragment (red circles) was monitored with a confocal fluorescence microscope as described¹⁷, ³⁰. The data were normalized to the background fluorescence. Error bars represent SEMs derived from three separate measurements. Fitting of the data to a dose-response curved yielded an EC50 of 12 nM for both the WT and mutant C₂AB fragment, which is comparable to earlier results obtained for WT C₂AB fragment³⁰. (b) The R398Q,R399Q mutation impairs the vesicle clustering activity of the C₂AB fragment. Mixtures of liposomes (100 nm average size) with WT or mutant C₂B domain or C₂AB fragment were prepared as described²⁷, and the change in particle size as a function of time was measured by DLS after addition of 100 μM Ca²⁺. For the experiment performed with the R398Q,R399Q mutant C₂AB fragment, 1 mM Ca²⁺ was added after 500 s.