Presynaptic active zone

Abstract:Summary Neurotransmitters are released at presynaptic active zones (AZs). In the fly Drosophila , monoclonal antibody (MAB) nc82 specifically labels AZs. We employ nc82 to identify Bruchpilot protein (BRP) as a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC, which binds RIM1 in a complex with Bassoon and Munc13-1. The C terminus of BRP displays structural similarities to multifunctional cytoskeletal proteins. During development, transcription of the bruchpilot locus ( brp ) coincides with neuronal differentiation. Panneural reduction of BRP expression by RNAi constructs permits a first functional characterization of this large AZ protein: larvae show reduced evoked but normal spontaneous transmission at neuromuscular junctions. In adults, we observe loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence " bruchpilot "—crash pilot). We propose that BRP is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila .

Abstract:Neurotransmitters are released by synaptic vesicle exocytosis at the active zone of a presynaptic nerve terminal. In this review, I discuss the molecular composition and function of the active zone. Active zones are composed of an evolutionarily conserved protein complex containing as core constituents RIM, Munc13, RIM-BP, α-liprin, and ELKS proteins. This complex docks and primes synaptic vesicles for exocytosis, recruits Ca 2+ channels to the site of exocytosis, and positions the active zone exactly opposite to postsynaptic specializations via transsynaptic cell-adhesion molecules. Moreover, this complex mediates short- and long-term plasticity in response to bursts of action potentials, thus critically contributing to the computational power of a synapse.

Abstract:The molecular organization of presynaptic active zones during calcium influx–triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) were entirely lost, Ca 2+ channels were reduced in density, evoked vesicle release was depressed, and short-term plasticity was altered. BRP-like proteins seem to establish proximity between Ca 2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity.

Abstract:Rab3 is a neuronal GTP-binding protein that regulates fusion of synaptic vesicles and is essential for long-term potentiation of hippocampal mossy fibre synapses1,2,3,4,5 More than thirty Rab GTP-binding proteins are known to function in diverse membrane transport pathways, although their mechanisms of action are unclear We have now identified a putative Rab3-effector protein called Rim Rim is composed of an amino-terminal zinc-finger motif and carboxy-terminal PDZ and C2 domains It binds only to GTP (but not to GDP)-complexed Rab3, and interacts with no other Rab protein tested There is enrichment of Rab3 and Rim in neurons, where they have complementary distributions Rab3 is found only on synaptic vesicles, whereas Rim is localized to presynaptic active zones in conventional synapses, and to presynaptic ribbons in ribbon synapses Transfection of PC12 cells with the amino-terminal domains of Rim greatly enhances regulated exocytosis in a Rab3-dependent manner We propose that Rim serves as a Rab3-dependent regulator of synaptic-vesicle fusion by forming a GTP-dependent complex between synaptic plasma membranes and docked synaptic vesicles

Abstract:Calcium ions serve as intracellular messengers in 2 activities of hair cells: in conjunction with Ca2(+)-activated K+ channels, they produce the electrical resonance that tunes each cell to a specific frequency of stimulation, and they trigger the release of a chemical synaptic transmitter. Our experiments indicate that both of these functions are conducted within a region that extends a few hundred nanometers around each presynaptic active zone. In focal electrical recordings from the plasma membranes of isolated anuran hair cells, we found nearly all of a cell's Ca2+ channels and Ca2(+)-activated K+ channels clumped at a fixed ratio in an average of 20 clusters on the basolateral membrane surface. Because serial-section electron microscopy indicated that each hair cell has approximately 19 afferent synaptic contacts with a similar distribution upon its basolateral surface, we conclude that the channel clusters coincide with synaptic active zones. Ensemble-variance analysis of current fluctuations indicated that each cell has a total of approximately 1800 Ca2+ channels and approximately 700 Ca2(+)- activated K+ channels; if these are uniformly divided, we estimate that each channel cluster contains approximately 90 Ca2+ and approximately 40 Ca2(+)-activated K+ channels. Freeze-fracture electron microscopy demonstrated an average of 133 large intramembrane particles in the presynaptic membrane at each active zone, an observation that suggests that the particles are the clustered channels. We used the K+ channel's sensitivity to intracellular Ca2+ to assay the concentration of free Ca2+ in the presynaptic cytoplasm, which we found to vary between 10 microM and 1 mM over the physiological range of membrane potentials. The inferred concentrations agreed with the values predicted for free diffusion of Ca2+ away from Ca2+ channels scattered randomly within a 300-nm-diameter synaptic active zone. The close association among Ca2+ channels, Ca2(+)-activated K+ channels, and synaptic active zones is necessary both for the rapid activation of K+ currents required in electrical resonance and for the transmission at afferent synapses of information about the phases of high-frequency stimuli.