Subunit composition, molecular environment, and activation of native TRPC channels encoded by their interactomes.

In the mammalian brain TRPC channels, a family of Ca2+-permeable cation channels, are involved in a variety of processes from neuronal growth and synapse formation to transmitter release, synaptic transmission and plasticity. The molecular appearance and operation of native TRPC channels, however, remained poorly understood. Here, we used high-resolution proteomics to show that TRPC channels in the rodent brain are macro-molecular complexes of more than 1 MDa in size that result from the co-assembly of the tetrameric channel core with an ensemble of interacting proteins (interactome). The core(s) of TRPC1-, C4-, and C5-containing channels are mostly heteromers with defined stoichiometries for each subtype, whereas TRPC3, C6, and C7 preferentially form homomers. In addition, TRPC1/C4/C5 channels may co-assemble with the metabotropic glutamate receptor mGluR1, thus guaranteeing both specificity and reliability of channel activation via the phospholipase-Ca2+ pathway. Our results unveil the subunit composition of native TRPC channels and resolve the molecular details underlying their activation.

Alix is required for activity-dependent bulk endocytosis at brain synapses.

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity-dependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations.

A synaptic temperature sensor for body cooling.

Deep brain temperature detection by hypothalamic warm-sensitive neurons (WSNs) has been proposed to provide feedback information relevant for thermoregulation. WSNs increase their action potential firing rates upon warming, a property that has been presumed to rely on the composition of thermosensitive ion channels within WSNs. Here, we describe a synaptic mechanism that regulates temperature sensitivity of preoptic WSNs and body temperature. Experimentally induced warming of the mouse hypothalamic preoptic area in vivo triggers body cooling. TRPM2 ion channels facilitate this homeostatic response and, at the cellular level, enhance temperature responses of WSNs, thereby linking WSN function with thermoregulation for the first time. Rather than acting within WSNs, we-unexpectedly-find TRPM2 to temperature-dependently increase synaptic drive onto WSNs by disinhibition. Our data emphasize a network-based interoceptive paradigm that likely plays a key role in encoding body temperature and that may facilitate integration of diverse inputs into thermoregulatory pathways.

Auxiliary Subunits Regulate the Dendritic Turnover of AMPA Receptors in Mouse Hippocampal Neurons.

Different families of auxiliary subunits regulate the function and trafficking of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the central nervous system. While a facilitatory role of auxiliary subunits in ER export and forward trafficking of newly synthesized AMPA receptors is firmly established, it is unclear whether auxiliary subunits also control endosomal receptor turnover in dendrites. Here, we manipulated the composition of AMPA receptor complexes in cultured hippocampal neurons by overexpression of two auxiliary subunits, transmembrane AMPAR regulatory protein (TARP) γ-8 or cysteine knot AMPAR-modulating protein (CKAMP) 44a, and monitored dendritic receptor cycling in live-cell imaging experiments. Receptor surface delivery was assayed using a modified AMPA receptor subunit carrying the pH-dependent fluorophore superecliptic pHluorin (SEP-GluA1), which regains its fluorescence during receptor exocytosis, when transiting from the acidic lumen of transport organelles to the neutral extracellular medium. Strikingly, we observed a dramatic reduction in the spontaneous fusion rate of AMPA receptor-containing organelles in neurons overexpressing either type of auxiliary subunit. An analysis of intracellular receptor distribution also revealed a decreased receptor pool in dendritic recycling endosomes, suggesting that incorporation of TARPγ-8 or CKAMP44a in receptor complexes generally diminishes cycling through the endosomal compartment. To directly analyze dendritic receptor turnover, we also generated a new reporter by N-terminal fusion of a self-labeling HaloTag to an AMPA receptor subunit (HaloTag-GluA1), which allows for selective, irreversible staining of surface receptors. Pulse chase-experiments with HaloTag-GluA1 indeed demonstrated that overexpression of TARPγ-8 or CKAMP44a reduces the constitutive internalization rate of surface receptors at extrasynaptic but not synaptic sites. Thus, our data point to a yet unrecognized regulatory function of TARPγ-8 and CKAMP44a, by which these structurally unrelated auxiliary subunits delay local recycling and increase surface lifetime of extrasynaptic AMPA receptors.

A short isoform of STIM1 confers frequency-dependent synaptic enhancement.

Store-operated Ca2+-entry (SOCE) regulates basal and receptor-triggered Ca2+ signaling with STIM proteins sensing the endoplasmic reticulum (ER) Ca2+ content and triggering Ca2+ entry by gating Orai channels. Although crucial for immune cells, STIM1's role in neuronal Ca2+ homeostasis is controversial. Here, we characterize a splice variant, STIM1B, which shows exclusive neuronal expression and protein content surpassing conventional STIM1 in cerebellum and of significant abundance in other brain regions. STIM1B expression results in a truncated protein with slower kinetics of ER-plasma membrane (PM) cluster formation and ICRAC, as well as reduced inactivation. In primary wild-type neurons, STIM1B is targeted by its spliced-in domain B to presynaptic sites where it converts classic synaptic depression into Ca2+- and Orai-dependent short-term synaptic enhancement (STE) at high-frequency stimulation (HFS). In conjunction with altered STIM1 splicing in human Alzheimer disease, our findings highlight STIM1 splicing as an important regulator of neuronal calcium homeostasis and of synaptic plasticity.

Synergistic actions of v-SNARE transmembrane domains and membrane-curvature modifying lipids in neurotransmitter release.

Vesicle fusion is mediated by assembly of SNARE proteins between opposing membranes. While previous work suggested an active role of SNARE transmembrane domains (TMDs) in promoting membrane merger (Dhara et al., 2016), the underlying mechanism remained elusive. Here, we show that naturally-occurring v-SNARE TMD variants differentially regulate fusion pore dynamics in mouse chromaffin cells, indicating TMD flexibility as a mechanistic determinant that facilitates transmitter release from differentially-sized vesicles. Membrane curvature-promoting phospholipids like lysophosphatidylcholine or oleic acid profoundly alter pore expansion and fully rescue the decelerated fusion kinetics of TMD-rigidifying VAMP2 mutants. Thus, v-SNARE TMDs and phospholipids cooperate in supporting membrane curvature at the fusion pore neck. Oppositely, slowing of pore kinetics by the SNARE-regulator complexin-2 withstands the curvature-driven speeding of fusion, indicating that pore evolution is tightly coupled to progressive SNARE complex formation. Collectively, TMD-mediated support of membrane curvature and SNARE force-generated membrane bending promote fusion pore formation and expansion.

TRPC channels regulate Ca2+-signaling and short-term plasticity of fast glutamatergic synapses.

Transient receptor potential (TRP) proteins form Ca2+-permeable, nonselective cation channels, but their role in neuronal Ca2+ homeostasis is elusive. In the present paper, we show that TRPC channels potently regulate synaptic plasticity by changing the presynaptic Ca2+-homeostasis of hippocampal neurons. Specifically, loss of TRPC1/C4/C5 channels decreases basal-evoked secretion, reduces the pool size of readily releasable vesicles, and accelerates synaptic depression during high-frequency stimulation (HFS). In contrast, primary TRPC5 channel-expressing neurons, identified by a novel TRPC5-τ-green fluorescent protein (τGFP) knockin mouse line, show strong short-term enhancement (STE) of synaptic signaling during HFS, indicating a key role of TRPC5 in short-term plasticity. Lentiviral expression of either TRPC1 or TRPC5 turns classic synaptic depression of wild-type neurons into STE, demonstrating that TRPCs are instrumental in regulating synaptic plasticity. Presynaptic Ca2+ imaging shows that TRPC activity strongly boosts synaptic Ca2+ dynamics, showing that TRPC channels provide an additional presynaptic Ca2+ entry pathway, which efficiently regulates synaptic strength and plasticity.

Large scale, unbiased analysis of elementary calcium signaling events in cardiac myocytes.

The identification of spatiotemporally restricted Ca2+ signals, Ca2+ sparks, was instrumental for our understanding of cardiac Ca2+ homeostasis. High-speed 2D confocal imaging enables acquisition of such Ca2+ sparks with high-content information but their full appreciation is constrained by the lack of unbiased and easy-to-use analysis tools. We developed a software toolset for unbiased and automatic Ca2+ spark analysis for huge data sets of subcellular Ca2+ signals. iSpark was developed to be scanner and detector independent. In myocytes from hearts subjected to various degrees of hypertrophy we acquired >5.000.000 Ca2+ sparks from 14 mice. The iSpark-enabled analysis of this large Ca2+ spark data set showed that the highly organized distribution of Ca2+ sparks present in healthy cells disarrayed concomitant with the development of aberrant transverse tubules and disease severity. Thus, iSpark represents a versatile and universal tool for analyzing local Ca2+ signaling in healthy as well as diseased, aberrant local Ca2+ signal transduction. The results from the unbiased analysis of large data sets provide a deeper insight into possible mechanisms contributing to the onset and progression of cardiac diseases such as hypertrophy.

The SNAP-25 linker supports fusion intermediates by local lipid interactions.

SNAP-25 is an essential component of SNARE complexes driving fast Ca2+-dependent exocytosis. Yet, the functional implications of the tandem-like structure of SNAP-25 are unclear. Here, we have investigated the mechanistic role of the acylated "linker" domain that concatenates the two SNARE motifs within SNAP-25. Refuting older concepts of an inert connector, our detailed structure-function analysis in murine chromaffin cells demonstrates that linker motifs play a crucial role in vesicle priming, triggering, and fusion pore expansion. Mechanistically, we identify two synergistic functions of the SNAP-25 linker: First, linker motifs support t-SNARE interactions and accelerate ternary complex assembly. Second, the acylated N-terminal linker segment engages in local lipid interactions that facilitate fusion triggering and pore evolution, putatively establishing a favorable membrane configuration by shielding phospholipid headgroups and affecting curvature. Hence, the linker is a functional part of the fusion complex that promotes secretion by SNARE interactions as well as concerted lipid interplay.

A mechanism for exocytotic arrest by the Complexin C-terminus.

ComplexinII (CpxII) inhibits non-synchronized vesicle fusion, but the underlying mechanisms have remained unclear. Here, we provide evidence that the far C-terminal domain (CTD) of CpxII interferes with SNARE assembly, thereby arresting tonic exocytosis. Acute infusion of a CTD-derived peptide into mouse chromaffin cells enhances synchronous release by diminishing premature vesicle fusion like full-length CpxII, indicating a direct, inhibitory function of the CTD that sets the magnitude of the primed vesicle pool. We describe a high degree of structural similarity between the CpxII CTD and the SNAP25-SN1 domain (C-terminal half) and show that the CTD peptide lowers the rate of SDS-resistant SNARE complex formation in vitro. Moreover, corresponding CpxII:SNAP25 chimeras do restore complexin's function and even 'superclamp' tonic secretion. Collectively, these results support a so far unrecognized clamping mechanism wherein the CpxII C-terminus hinders spontaneous SNARE complex assembly, enabling the build-up of a release-ready pool of vesicles for synchronized Ca2+-triggered exocytosis.

v-SNARE function in chromaffin cells.

Vesicle fusion is elementary for intracellular trafficking and release of signal molecules, thus providing the basis for diverse forms of intercellular communication like hormonal regulation or synaptic transmission. A detailed characterization of the mechanisms underlying exocytosis is key to understand how the nervous system integrates information and generates appropriate responses to stimuli. The machinery for vesicular release employs common molecular players in different model systems including neuronal and neuroendocrine cells, in particular members of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family, Sec1/Munc18-like proteins, and other accessory factors. To achieve temporal precision and speed, excitable cells utilize specialized regulatory proteins like synaptotagmin and complexin, whose interplay putatively synchronizes vesicle fusion and enhances stimulus-secretion coupling. In this review, we aim to highlight recent progress and emerging views on the molecular mechanisms, by which constitutively forming SNAREpins are organized in functional, tightly regulated units for synchronized release. Specifically, we will focus on the role of vesicle associated membrane proteins, also referred to as vesicular SNAREs, in fusion and rapid cargo discharge. We will further discuss the functions of SNARE regulators during exocytosis and focus on chromaffin cell as a model system of choice that allows for detailed structure-function analyses and direct measurements of vesicle fusion under precise control of intracellular [Ca]i.

Astrocytes control synaptic strength by two distinct v-SNARE-dependent release pathways.

Communication between glia cells and neurons is crucial for brain functions, but the molecular mechanisms and functional consequences of gliotransmission remain enigmatic. Here we report that astrocytes express synaptobrevin II and cellubrevin as functionally non-overlapping vesicular SNARE proteins on glutamatergic vesicles and neuropeptide Y-containing large dense-core vesicles, respectively. Using individual null-mutants for Vamp2 (synaptobrevin II) and Vamp3 (cellubrevin), as well as the corresponding compound null-mutant for genes encoding both v-SNARE proteins, we delineate previously unrecognized individual v-SNARE dependencies of astrocytic release processes and their functional impact on neuronal signaling. Specifically, we show that astroglial cellubrevin-dependent neuropeptide Y secretion diminishes synaptic signaling, while synaptobrevin II-dependent glutamate release from astrocytes enhances synaptic signaling. Our experiments thereby uncover the molecular mechanisms of two distinct v-SNARE-dependent astrocytic release pathways that oppositely control synaptic strength at presynaptic sites, elucidating new avenues of communication between astrocytes and neurons.

Heteromeric channels formed by TRPC1, TRPC4 and TRPC5 define hippocampal synaptic transmission and working memory.

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high-resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from Trpc1/Trpc4/Trpc5-triple-knockout (Trpc1/4/5-/-) mice, lacking any TRPC1-, TRPC4-, or TRPC5-containing channels, action potential-triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. In vivo, Trpc1/4/5-/- mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5-/- animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.

v-SNARE transmembrane domains function as catalysts for vesicle fusion.

Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca(2+)-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle's outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.

Synaptobrevin transmembrane domain determines the structure and dynamics of the SNARE motif and the linker region.

The vesicular protein synaptobrevin II (sybII) constitutes a central component of the SNARE complex, which mediates vesicle fusion in neuronal exocytosis. Previous studies revealed that the transmembrane domain (TMD) of sybII is playing a critical role in the fusion process and is involved in all distinct fusion stages from priming to fusion pore opening. Here, we analyzed sequence-dependent effects of sybII and of mutants of sybII on both structure and flexibility of the protein and the interactions with a phospholipid bilayer by means of microsecond atomistic simulations. The sybII TMD was found to direct the folding of both the juxtamembrane helix and of the connecting linker and thus to influence both the intrinsic helicity and flexibility. Fusion active peptides revealed two helical segments, one for the juxtamembrane region and one for the TMD, connected by a flexible linker. In contrast, a fusion-inactive poly-leucine TMD mutant assumes a structure with a comparably rigid linker that is suggested to hinder the formation of the trans-SNARE complex during fusion. Kinking of the TMD at the central glycine together with anchoring of the TMD via conserved tryptophans and a lysine in position 94 likely yields an enhanced flexibility of sybII for different membrane thickness. All studied peptides were found to deform the outer membrane layer by altering the lipid head group orientation, causing partial membrane dehydration and enhancing lipid protrusions. These effects weaken the integrity of the outer membrane layer and are attributed mainly to the highly charged linker and JM regions of sybII.

Complexins: small but capable.

Despite intensive research, it is still unclear how an immediate and profound acceleration of exocytosis is triggered by appropriate Ca(2+)-stimuli in presynaptic terminals. This is due to the fact that the molecular mechanisms of "docking" and "priming" reactions, which set up secretory vesicles to fuse at millisecond time scale, are extremely hard to study. Yet, driven by a fruitful combination of in vitro and in vivo analyses, our mechanistic understanding of Ca(2+)-triggered vesicle fusion has certainly advanced in the past few years. In this review, we aim to highlight recent progress and emerging views on the molecular mechanisms, by which constitutively forming SNAREpins are organized in functional, tightly regulated units for synchronized release. In particular, we will focus on the role of the small regulatory factor complexin whose function in Ca(2+)-dependent exocytosis has been controversially discussed for more than a decade. Special emphasis will also be laid on the functional relationship of complexin and synaptotagmin, as both proteins possibly act as allies and/or antagonists to govern SNARE-mediated exocytosis.

A structural role for the synaptobrevin 2 transmembrane domain in dense-core vesicle fusion pores.

Ca(2+)-triggered release of neurotransmitters and hormones depends on soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to drive the fusion of the vesicle and plasma membranes. The formation of the SNARE complex by the vesicle SNARE synaptobrevin 2 (syb2) and the two plasma membrane SNAREs syntaxin (syx) and SNAP-25 draws the two membranes together, but the events that follow membrane juxtaposition, and the ways that SNAREs remodel lipid membranes remain poorly understood. The SNAREs syx and syb2 have transmembrane domains (TMDs) that can exert force directly on the lipid bilayers. The TMD of syx influences fusion pore flux in a manner that suggests it lines the nascent fusion pore through the plasma membrane. The TMD of syb2 traverses the vesicle membrane and is the most likely partner to syx in completing a proteinaceous fusion pore through the vesicle membrane, but the role of this vesicle SNARE in fusion pores has yet to be tested. Here amperometry and conductance measurements were performed to probe the function of the syb2 TMD in fusion pores formed during catecholamine exocytosis in mouse chromaffin cells. Fusion pore flux was sensitive to the size and charge of TMD residues near the N terminus; fusion pore conductance was altered by substitutions at these sites. Unlike syx, the syb2 residues that influence fusion pore permeation fell along two α-helical faces of its TMD, rather than one. These results indicate a role for the syb2 TMD in nascent fusion pores, but in a very different structural arrangement from that of the syx TMD.

Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics.

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca(2+)-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca(2+) concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca(2+)-triggered exocytosis by increasing the Ca(2+) affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca(2+)-triggered release apparatus.

Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering.

SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

Synaptobrevin2 is the v-SNARE required for cytotoxic T-lymphocyte lytic granule fusion.

Cytotoxic T lymphocytes kill virus-infected and tumorigenic target cells through the release of perforin and granzymes via fusion of lytic granules at the contact site, the immunological synapse. It has been postulated that this fusion process is mediated by non-neuronal members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex protein family. Here, using a synaptobrevin2-monomeric red fluorescence protein knock-in mouse we demonstrate that, surprisingly, the major neuronal v-SNARE synaptobrevin2 is expressed in cytotoxic T lymphocytes and exclusively localized on granzyme B-containing lytic granules. Cleavage of synaptobrevin2 by tetanus toxin or ablation of the synaptobrevin2 gene leads to a complete block of lytic granule exocytosis while leaving upstream events unaffected, identifying synaptobrevin2 as the v-SNARE responsible for the fusion of lytic granules at the immunological synapse.