Dieter Bruns

SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol‐dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol‐dependent membrane rafts since they are Triton X‐100‐soluble and do not co‐patch with raft markers. Synaptosomal‐associated protein (SNAP)‐25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.

v‐SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF‐attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle‐associated SNARE proteins (v‐SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca2+‐dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v‐SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v‐SNAREs amino terminus regulates the vesicles primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v‐SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

A Structural Change Occurs upon Binding of Syntaxin to SNAP-25

The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work demonstrated that syntaxin and SNAP-25 bind to each other with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used here to study the α-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displayed approximately 43% α-helical content. In contrast, the α-helical content of SNAP-25 was low under physiological conditions. Formation of the SNAP-25-syntaxin complex was associated with a dramatic increase in α-helicity. Interaction of a 90-residue NH2-terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain lead to a similar but less pronounced increase in α-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolished the induced structural change and disrupted the interaction monitored by binding assays. Replacements with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.

Quantal Release of Serotonin

We have studied the origin of quantal variability for small synaptic vesicles (SSVs) and large dense-cored vesicles (LDCVs). As a model, we used serotonergic Retzius neurons of leech that allow for combined amperometrical and morphological analyses of quantal transmitter release. We find that the transmitter amount released by a SSV varies proportionally to the volume of the vesicle, suggesting that serotonin is stored at a constant intravesicular concentration and is completely discharged during exocytosis. Transmitter discharge from LDCVs shows a higher degree of variability than is expected from their size distribution, and bulk release from LDCVs is slower than release from SSVs. On average, differences in the transmitter amount released from SSVs and LDCVs are proportional to the size differences of the organelles, suggesting that transmitter is stored at similar concentrations in SSVs and LDCVs.

Synaptobrevin N-terminally bound to syntaxin–SNAP-25 defines the primed vesicle state in regulated exocytosis

Time-resolved measurements of exocytosis identify a domain of the SNARE complex required to keep vesicles readily releasable.

CAPS1 Regulates Catecholamine Loading of Large Dense-Core Vesicles

CAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs.

A fast activating presynaptic reuptake current during serotonergic transmission in identified neurons of Hirudo

An electrogenic serotonin (5-HT) uptake process was characterized in the serotonergic Retzius-P cell synapse of the leech, and the simultaneous activation of this presynaptic reuptake and the postsynaptic response was monitored during evoked transmitter release. A presynaptic, Na(+)-dependent inward current upon application of 5-HT was isolated at membrane potentials between -80 and +60 mV. Its identification as a transmitter uptake current was confirmed by monitoring accumulation of the autofluorescent 5-HT analog 5,7-dihydroxytryptamine during activation of this current. To study the kinetics of 5-HT reuptake in functional synapses, transmitter release was stimulated by flash photolysis of the Ca(2+)-caging DM-nitrophen. The results demonstrate that reuptake activates with a minimal delay of less than a millisecond during synaptic transmission. It acts as a rapid transmitter removal system to determine the time course of the postsynaptic response and monitors the kinetics of transmitter clearance at the synaptic site.

TRPC5 Is a Ca2+-activated Channel Functionally Coupled to Ca2+-selective Ion Channels

TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca2+ in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca2+ concentrations (Ca2+i) revealed a dose-dependent activation of TRPC5 channels by internal Ca2+ with EC50 of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca2+i induced by photolysis of caged Ca2+ showed that the Ca2+ activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 ± 0.2 ms at Ca2+i below 10 μm, suggesting that the action of internal Ca2+ is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 ± 0.1 s was also observed at Ca2+i above 10 μm. In support of a Ca2+-activation mechanism, the thapsigargin-induced release of Ca2+ from internal stores activated TRPC5 channels transiently, and the subsequent Ca2+ entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the α1C and β2 subunits of L-type Ca2+ channels, we found that Ca2+ entry through either calcium-release-activated-calcium or voltage-dependent Ca2+ channels is sufficient for TRPC5 channel activation. The Ca2+ entry activated TRPC5 channels under buffering of internal Ca2+ with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca2+-activated cation channels that are functionally coupled to Ca2+-selective ion channels through local Ca2+ increases beneath the plasma membrane.

CAPS Facilitates Filling of the Rapidly Releasable Pool of Large Dense-Core Vesicles

Calcium-activator protein for secretion (CAPS) is a cytosolic protein that associates with large dense-core vesicles and is involved in their secretion. Mammals express two CAPS isoforms, which share a similar domain structure including a Munc13 homology domain that is believed to be involved in the priming of secretory vesicles. A variety of studies designed to perturb CAPS function indicate that CAPS is involved in the secretion of large dense-core vesicles, but where in the secretory pathway CAPS acts is still under debate. Mice in which one allele of the CAPS-1 gene is deleted exhibit a deficit in catecholamine secretion from chromaffin cells. We have examined catecholamine secretion from chromaffin cells in which both CAPS genes were deleted and show that the deletion of both CAPS isoforms causes a strong reduction in the pool of rapidly releasable chromaffin granules and of sustained release during ongoing stimulation. We conclude that CAPS is required for the adequate refilling and/or maintenance of a rapidly releasable granule pool.

Molecular determinants of exocytosis.

Membrane fusion processes occur throughout the cell. Among those, exocytosis of secretory organelles is probably one of the fastest fusion events animal cells can achieve. Structural and functional studies have provided a conceptual framework and a starting point for our mechanistic understanding of how SNARE proteins may contribute to the final step in exocytosis.