Voltage-dependent Ca(2+) channels (CaVs) represent the principal source of Ca(2+) ions that trigger evoked neurotransmitter release from presynaptic boutons. Ca(2+) influx is mediated mainly via CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels, which differ in their properties. Their relative contribution to synaptic transmission changes during development and tunes neurotransmission during synaptic plasticity. The mechanism of differential recruitment of CaV2.1 and CaV2.2 to release sites is largely unknown. Here, we show that the presynaptic scaffolding protein Bassoon localizes specifically CaV2.1 to active zones via molecular interaction with the RIM-binding proteins (RBPs). A genetic deletion of Bassoon or an acute interference with Bassoon-RBP interaction reduces synaptic abundance of CaV2.1, weakens P/Q-type Ca(2+) current-driven synaptic transmission, and results in higher relative contribution of neurotransmission dependent on CaV2.2. These data establish Bassoon as a major regulator of the molecular composition of the presynaptic neurotransmitter release sites.

Unravelling principles underlying neurotransmitter release are key to understand neural signaling. Here, we describe how surface mobility of voltage-dependent calcium channels (VDCCs) modulates release probabilities (P(r)) of synaptic vesicles (SVs). Coupling distances of <10 to >100 nm have been reported for SVs and VDCCs in different synapses. Tracking individual VDCCs revealed that within hippocampal synapses, ∼60% of VDCCs are mobile while confined to presynaptic membrane compartments. Intracellular Ca(2+) chelation decreased VDCC mobility. Increasing VDCC surface populations by co-expression of the α2δ1 subunit did not alter channel mobility but led to enlarged active zones (AZs) rather than higher channel densities. VDCCs thus scale presynaptic scaffolds to maintain local mobility. We propose that dynamic coupling based on mobile VDCCs supports calcium domain cooperativity and tunes neurotransmitter release by equalizing Pr for docked SVs within AZs.

Synapses depend on trafficking of key membrane proteins by lateral diffusion from surface populations and by exocytosis from intracellular pools. The cell adhesion molecule neurexin (Nrxn) plays essential roles in synapses, but the dynamics and regulation of its trafficking are unknown. Here, we performed single-particle tracking and live imaging of transfected, epitope-tagged Nrxn variants in cultured rat and mouse wild-type or knock-out neurons. We observed that structurally larger ␣Nrxn molecules are more mobile in the plasma membrane than smaller ␤Nrxns because ␣Nrxns displayed higher diffusion coefficients in extrasynaptic regions and excitatory or inhibitory terminals. We found that well characterized interactions with extracellular binding partners regulate the surface mobility of Nrxns. Binding to neurexophilin-1 (Nxph1) reduced the surface diffusion of ␣Nrxns when both molecules were coexpressed. Conversely, impeding other interactions by insertion of splice sequence #4 or removal of extracellular Ca 2ϩ augmented the mobility of ␣Nrxns and ␤Nrxns. We also determined that fast axonal transport delivers Nrxns to the neuronal surface because Nrxns comigrate as cargo on synaptic vesicle protein transport vesicles (STVs). Unlike surface mobility, intracellular transport of ␤Nrxn ϩ STVs was faster than that of ␣Nrxns, but both depended on the microtubule motor protein KIF1A and neuronal activity regulated the velocity. Large spontaneous fusion of Nrxn ϩ STVs occurred simultaneously with synaptophysin on axonal membranes mostly outside of active presynaptic terminals. Surface Nrxns enriched at synaptic terminals where ␣Nrxns and Nxph1/␣Nrxns recruited GABA A R subunits. Therefore, our results identify regulated dynamic trafficking as an important property of Nrxns that corroborates their function at synapses.

Biological system is unique in that component biomolecules are self-organized and emerge adaptable, stable and energy-saving functions. To understand the essential condition for reproducing the properties and constructing stable system from unstable flexible components, we are designing artificial muscle using myosin and DNA nanostructure. System size, spatial positioning, degree of intrinsic noise and the mechanical communication in the synthesized muscle can be modulated and we'll observe the internal dynamics and the system behavior at the single molecule resolution. We have developed several novel tools to modulate the system and monitor the internal dynamics with high precision.