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?2? expression sets presynaptic calcium channel abundance and release probability.

ABSTRACT: Synaptic neurotransmitter release is driven by Ca(2+) influx through active zone voltage-gated calcium channels (VGCCs). Control of active zone VGCC abundance and function remains poorly understood. Here we show that a trafficking step probably sets synaptic VGCC levels in rats, because overexpression of the pore-forming ?1(A) VGCC subunit fails to change synaptic VGCC abundance or function. ?2?s are a family of glycosylphosphatidylinositol (GPI)-anchored VGCC-associated subunits that, in addition to being the target of the potent neuropathic analgesics gabapentin and pregabalin (?2?-1 and ?2?-2), were also identified in a forward genetic screen for pain genes (?2?-3). We show that these proteins confer powerful modulation of presynaptic function through two distinct molecular mechanisms. First, ?2? subunits set synaptic VGCC abundance, as predicted from their chaperone-like function when expressed in non-neuronal cells. Second, ?2?s configure synaptic VGCCs to drive exocytosis through an extracellular metal ion-dependent adhesion site (MIDAS), a conserved set of amino acids within the predicted von Willebrand A domain of ?2?. Expression of ?2? with an intact MIDAS motif leads to an 80% increase in release probability, while simultaneously protecting exocytosis from blockade by an intracellular Ca(2+) chelator. ?2?s harbouring MIDAS site mutations still drive synaptic accumulation of VGCCs; however, they no longer change release probability or sensitivity to intracellular Ca(2+) chelators. Our data reveal dual functionality of these clinically important VGCC subunits, allowing synapses to make more efficient use of Ca(2+) entry to drive neurotransmitter release.

SUBMITTER: Hoppa MB

PROVIDER: S-EPMC3376018 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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α2δ expression sets presynaptic calcium channel abundance and release probability.

Hoppa Michael B MB Lana Beatrice B Margas Wojciech W Dolphin Annette C AC Ryan Timothy A TA

Nature 20120513 7401

Synaptic neurotransmitter release is driven by Ca(2+) influx through active zone voltage-gated calcium channels (VGCCs). Control of active zone VGCC abundance and function remains poorly understood. Here we show that a trafficking step probably sets synaptic VGCC levels in rats, because overexpression of the pore-forming α1(A) VGCC subunit fails to change synaptic VGCC abundance or function. α2δs are a family of glycosylphosphatidylinositol (GPI)-anchored VGCC-associated subunits that, in additi ...[more]

PMID: 22678293

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Project description:Presynaptic α2δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α2δ-2 can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ-2 is essential for the trans-synaptic mechanism. The novel function of α2δ-2 identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α2δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α2δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α2δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.

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.

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?2? expression sets presynaptic calcium channel abundance and release probability.

Publications

α2δ expression sets presynaptic calcium channel abundance and release probability.

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