Active zone core proteins in interaction with synaptic Ca2+ channels

• Applicant: Professor Dr. Stephan J. Sigrist
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2012 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 230148358

At the presynaptic active zone, Ca2+ channels trigger the release of synaptic vesicles (SVs) to transmit and transform neuronal information. Active zones are now known to be composed of an evolutionarily conserved complex containing as primary constituents RIM, Munc13, RIM-binding protein (RBP), alpha-liprin, and ELKS proteins. RIM and RBP directly interact directly with the Ca2+ channel intracellular C-termini using highly conserved motifs. Same time, physiological analysis suggests that Ca2+ channels get into very close physical distance from Ca2+ sensors of the SVs (10-100 nm). This nanodomain coupling might increase the efficacy, speed of synaptic transmission and be important for information processing and coding. Mechanisms of active zone protein function and nanodomain coupling, however, remain largely enigmatic. Our recent work at Drosophila NMJ model synapses provided evidence that loss of either RBP or ELKS-family protein Bruchpilot (BRP) results in i) structural deficits at active zones including a loss of Ca2+-channels ii) deficits in base-line transmission iii) severe loss of SV release probability associated with atypical short-term facilitation. Whether these phenotypes reflect defects in defining physical proximity or in the assembly of biochemical scaffolds facilitating Ca2+ triggered SV release remained open. The Drosophila alpha-1 subunit Cacophony (Cac) is the only representative of the N-/ P/Q-type family and solely responsible for SV release at NMJ synapses. We here propose to systematically explore the role of protein interactions between the intracellular domains of Cac and active zone proteins. We start interfering with specific Cac interactions by introducing suitable point mutations into a large point mutated cac genomic constructs, which will be subjected to detailed electrophysiological analysis (SV release probability, numbers of releasable SVs, and short- term plasticity indicative of nanodomain coupling). The active zone protein architecture might directly define SV binding and release sites (slots) in tight proximity to the channels. This hypothesis we seek to test on ultrastructural level (acute Channelrhodopsin-mediated stimulation followed by electron microscopic tomography). Last not least, we seek to address whether direct contacts between Cac/RBP and the SV release machinery components (particularly unc-13 family) are important for effective SV release as well. Collectively, by focusing on discrete interaction surfaces we hope to genetically uncouple assembly from truly functional deficits at active zones, and gain generic insights into the functional principles of the active zone core.

• DFG Programme: Research Grants