Dataset Information

Molecular Characterization of an SV Capture Site in the Mid-Region of the Presynaptic CaV2.1 Calcium Channel C-Terminal.

ABSTRACT: Neurotransmitter is released from presynaptic nerve terminals at fast-transmitting synapses by the action potential-gating of voltage dependent calcium channels (CaV), primarily of the CaV2.1 and CaV2.2 types. Entering Ca2+ diffuses to a nearby calcium sensor associated with a docked synaptic vesicle (SV) and initiates its fusion and discharge. Our previous findings that single CaVs can gate SV fusion argued for one or more tethers linking CaVs to docked SVs but the molecular nature of these tethers have not been established. We recently developed a cell-free, in vitro biochemical assay, termed SV pull-down (SV-PD), to test for SV binding proteins and used this to demonstrate that CaV2.2 or the distal third of its C-terminal can capture SVs. In subsequent reports we identified the binding site and characterized an SV binding motif. In this study, we set out to test if a similar SV-binding mechanism exists in the primary presynaptic channel type, CaV2.1. We cloned the chick variant of this channel and to our surprise found that it lacked the terminal third of the C-terminal, ruling out direct correlation with CaV2.2. We used SV-PD to identify an SV binding site in the distal half of the CaV2.1 C-terminal, a region that corresponds to the central third of the CaV2.2 C-terminal. Mutant fusion proteins combined with motif-blocking peptide strategies identified two domains that could account for SV binding; one in an alternatively spliced region (E44) and a second more distal site. Our findings provide a molecular basis for CaV2.1 SV binding that can account for recent evidence of C-terminal-dependent transmitter release modulation and that may contribute to SV tethering within the CaV2.1 single channel Ca2+ domain.

SUBMITTER: Snidal CA

PROVIDER: S-EPMC5958201 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Molecular Characterization of an SV Capture Site in the Mid-Region of the Presynaptic CaV2.1 Calcium Channel C-Terminal.

Snidal Christine A CA Li Qi Q Elliott Brittany B BB Mah Henry K-H HK Chen Robert H C RHC Gardezi Sabiha R SR Stanley Elise F EF

Frontiers in cellular neuroscience 20180511

Neurotransmitter is released from presynaptic nerve terminals at fast-transmitting synapses by the action potential-gating of voltage dependent calcium channels (CaV), primarily of the CaV2.1 and CaV2.2 types. Entering Ca<sup>2+</sup> diffuses to a nearby calcium sensor associated with a docked synaptic vesicle (SV) and initiates its fusion and discharge. Our previous findings that single CaVs can gate SV fusion argued for one or more tethers linking CaVs to docked SVs but the molecular nature o ...[more]

PMID: 29867360

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Project description:Efficient and reliable neurotransmission requires precise coupling between action potentials (APs), Ca2+ entry and neurotransmitter release. However, Ca2+ requirements for release, including the number of channels required, their subtypes, and their location with respect to primed vesicles, remains to be precisely defined for central synapses. Indeed, Ca2+ entry may occur through small numbers or even single open Ca2+ channels, but these questions remain largely unexplored in simple active zone (AZ) synapses common in the nervous system, and key to addressing Ca2+ channel and synaptic dysfunction underlying numerous neurologic and neuropsychiatric disorders. Here, we present single channel analysis of evoked AZ Ca2+ entry, using cell-attached patch clamp and lattice light-sheet microscopy (LLSM), resolving small channel numbers evoking Ca2+ entry following depolarization, at single AZs in individual central lamprey reticulospinal presynaptic terminals from male and females. We show a small pool (mean of 23) of Ca2+ channels at each terminal, comprising N-(CaV2.2), P/Q-(CaV2.1), and R-(CaV2.3) subtypes, available to gate neurotransmitter release. Significantly, of this pool only one to seven channels (mean of 4) open on depolarization. High temporal fidelity lattice light-sheet imaging reveals AP-evoked Ca2+ transients exhibiting quantal amplitude variations of 0-6 event sizes between individual APs and stochastic variation of precise locations of Ca2+ entry within the AZ. Further, total Ca2+ channel numbers at each AZ correlate to the number of presynaptic primed synaptic vesicles. Dispersion of channel openings across the AZ and the similar number of primed vesicles and channels indicate that Ca2+ entry via as few as one channel may trigger neurotransmitter release.SIGNIFICANCE STATEMENT Presynaptic Ca2+ entry through voltage-gated calcium channels (VGCCs) causes neurotransmitter release. To understand neurotransmission, its modulation, and plasticity, we must quantify Ca2+ entry and its relationship to vesicle fusion. This requires direct recordings from active zones (AZs), previously possible only at calyceal terminals containing many AZs, where few channels open following action potentials (APs; Sheng et al., 2012), and even single channel openings may trigger release (Stanley, 1991, 1993). However, recording from more conventional terminals with single AZs commonly found centrally has thus far been impossible. We addressed this by cell-attached recordings from acutely dissociated single lamprey giant axon AZs, and by lattice light sheet microscopy of presynaptic Ca2+ entry. We demonstrate nanodomains of presynaptic VGCCs coupling with primed vesicles with 1:1 stoichiometry.

Project description:In neurons, entry of extracellular calcium (Ca(2+)) into synaptic terminals through Cav2.1 (P/Q-type) Ca(2+) channels is the driving force for exocytosis of neurotransmitter-containing synaptic vesicles. This class of Ca(2+) channel is, therefore, pivotal during normal neurotransmission in higher organisms. In response to channel opening and Ca(2+) influx, specific Ca(2+)-binding proteins associate with cytoplasmic regulatory domains of the P/Q channel to modulate subsequent channel opening. Channel modulation in this way influences synaptic plasticity with consequences for higher-level processes such as learning and memory acquisition. The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) regulates the activity of all types of mammalian voltage-gated Ca(2+) channels, including the P/Q class, by direct binding to specific regulatory motifs. More recently, experimental evidence has highlighted a role for additional Ca(2+)-binding proteins, particularly of the CaBP and NCS families in the regulation of P/Q channels. NCS-1 is a protein found from yeast to humans and that regulates a diverse number of cellular functions. Physiological and genetic evidence indicates that NCS-1 regulates P/Q channel activity, including calcium-dependent facilitation, although a direct physical association between the proteins has yet to be demonstrated. In this study, we aimed to determine if there is a direct interaction between NCS-1 and the C-terminal cytoplasmic tail of the Cav2.1 α-subunit. Using distinct but complementary approaches, including in vitro binding of bacterially expressed recombinant proteins, fluorescence spectrophotometry, isothermal titration calorimetry, nuclear magnetic resonance, and expression of fluorescently tagged proteins in mammalian cells, we show direct binding and demonstrate that CaM can compete for it. We speculate about how NCS-1/Cav2.1 association might add to the complexity of calcium channel regulation mediated by other known calcium-sensing proteins and how this might help to fine-tune neurotransmission in the mammalian central nervous system.

Dataset Information

Molecular Characterization of an SV Capture Site in the Mid-Region of the Presynaptic CaV2.1 Calcium Channel C-Terminal.

Publications

Molecular Characterization of an SV Capture Site in the Mid-Region of the Presynaptic CaV2.1 Calcium Channel C-Terminal.

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