Studying Calmodulin / Munc Interaction

The efficacy of synaptic transmission between neurons can be transiently altered during neuronal network activity. This phenomenon of short-term plasticity (STP) is a key determinant of network properties, is involved in many physiological processes such as motor control, sound localization, or sensory adaptation, and is critically dependent on cytosolic calcium concentration. Due to their essential function in synaptic vesicle priming and in the modulation of synaptic strength, Munc13 proteins are key regulators of presynaptic short-term plasticity. However, the underlying molecular mechanisms and the identity of the calcium sensor/effector complexes involved are unclear. A conserved calmodulin (CaM) binding site was identified in UNC-13/Munc13 proteins. Chemical cross-linking experiments will be conducted between CaM and Munc13 using amine-reactive as well as photo-reactive cross-linkers and trifunctional reagents possessing a biotin label for enrichment of cross-linked products. After the cross-linking reaction, the cross-linked complexes will be cleaved by proteases and the created peptide mixtures will by analyzed by offline nano-HPLC / MALDI-TOF/TOF and online nano-HPLC / nano-ESI-Orbitrap mass spectrometry.