Synaptic transmission relies on an exquisitely orchestrated series of protein-protein interactions. Here we show that fusion driven by neuronal SNAREs is inhibited by the regulatory protein complexin. Furthermore, inner-leaflet mixing is strongly impaired relative to total lipid mixing, indicating that inhibition by complexin arrests fusion at hemifusion. When the calcium sensor synaptotagmin is added in the presence of calcium, inhibition by complexin is relieved and full fusion rapidly proceeds.

Ca2+-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca2+ wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca2+ and Ca2+-sensing fusion effectors in neurotransmitter release.

Synaptic transmission requires the controlled release of neurotransmitter from synaptic vesicles by membrane fusion with the presynaptic plasma membrane. SNAREs are the core constituents of the protein machinery responsible for synaptic membrane fusion. The mechanism by which SNAREs drive membrane fusion is thought to involve a hemifusion intermediate, a condition in which the outer leaflets of two bilayers are combined and the inner leaflets remain intact; however, hemifusion has been observed only as an end point rather than as an intermediate. Here, we examined the kinetics of membrane fusion of liposomes mediated by recombinant neuronal SNAREs using fluorescence assays that monitor both total lipid mixing and inner leaflet mixing. Our results demonstrate that hemifusion is dominant at the early stage of the fusion reaction. Over time, hemifusion transitioned to complete fusion, showing that hemifusion is a true intermediate. We also show that hemifusion intermediates can be trapped, likely as unproductive outcomes, by modulating the surface concentration of the SNARE proteins.In the neuron, SNARE 1 assembly plays a critical role in promoting the fusion of the synaptic vesicles with the plasma membrane (1-7). Cognate SNAREs pair to form a coiled coil structure that bridges two membranes (8, 9). The subsequent steps yielding one common phospholipid bilayer remain a matter of debate. It has been proposed that SNAREs involved in neurotransmitter release at synapses may promote membrane fusion by the formation of two juxtaposed transmembrane pores preassembled by the transmembrane domains of SNAREs in respective membranes (10). In sharp contrast, recent evidence for SNAREs involved in trafficking in yeast has indicated that hemifusion might be involved in the SNARE fusion pathway (11, 12), analogous to the lipid-protein stalk model generally accepted for viral membrane fusion proteins (13-17). However, hemifusion has been observed only as an outcome rather than as an intermediate, raising some concerns as to whether hemifusion is an off-pathway product in SNAREmediated membrane fusion (16). Alternatively, the mechanism by which neuronal SNAREs induce membrane fusion might be entirely different from those for other systems including yeast SNAREs.In this work, we examined the kinetics of membrane fusion of liposomes mediated by neuronal SNAREs syntaxin 1A, SNAP-25, and synaptobrevin using fluorescence assays (18) that monitored both total lipid mixing and inner leaflet mixing. Our results demonstrate that hemifusion is the main event at the early stage of the fusion reaction. Over time, hemifusion converts to the complete fusion, supporting strongly the theory that hemifusion is a true fusion intermediate. MATERIALS AND METHODSProtein Sample Preparation-Plasmid construction, protein expression, and purification for neuronal SNAREs were described elsewhere (19). Briefly, vesicle-associated (v-) SNARE synaptobrevin (amino acids 1-116) and a truncated version of target membrane (t-) SNARE syntaxin (amino acids 168 -28...

Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex facilitates intracellular membrane fusion. A single SNARE complex is thought to be insufficient; multiple copies of SNARE complexes must work cooperatively. However, the mechanism by which such a higher-order SNARE protein structure is assembled is unknown. EPR and fluorescence analyses show that at least three copies of target-membrane SNARE proteins self-assemble through the interaction between the transmembrane domains (TMDs), and this multimeric structure serves as scaffolding for trans-SNARE assembly. SNARE core formation in solution induces oligomerization of the TMDs of vesicle-associated SNAREs in the apposing membrane, transiently forming a supramolecular protein structure spanning two membranes. This higher-order protein intermediate evolves, by involving lipid molecules, to the hemifusion state. Hemifusion is subsequently followed by distal leaflet mixing and formation of the cis-SNARE complex.

Reversible fixation-release of CO 2 has attracted much attention during the last decades due to the economic and environmental benefits arising from the utilization of renewable resources and the growing concern on the greenhouse effect. 1-3 Numerous systems based on liquid primary or secondary amines have been developed for this process, in which CO 2 is chemically converted into ammonium carbamates or zwitterionic adducts at ambient temperature 4-6 and the fixed CO 2 is released upon heating. 7,8 Likewise, amino-functionalized synthetic polymers or mesoporous materials were also proved to be efficient in CO 2 capture. 9-11 Notably, ethylenimine functionalized mesoporous molecular sieve MCM-41 has higher absorption capacity than either pure polyethylenimine or MCM-41 alone and could be used as highly CO 2 -selective adsorbent for gas mixtures without the preremoval of moisture. 10 Endo and co-workers reported a new type of reversible CO 2 fixation by amidine derivatives and by polymers bearing an amidine moiety both in solution and solid state. 12 The cyclic amidine-functionalized copolymers exhibited better ability to retain CO 2 than corresponding low-molecular weight amidine at ambient temperature and could be applied to reversible fixation-release of CO 2 . The CO 2 fixation by polymers in the solid state may be one of the most simple, economic and effective methods for CO 2 recovery, though the fixing efficiency is relatively low.During the past decade, N-heterocyclic carbenes were studied extensively as versatile ligands 13 and effective organocatalysts. 14 Because of their high basicity, 15 N-heterocyclic carbenes can react rapidly with CO 2 to afford zwitterionic adducts (designated as NHC-CO 2 ), even at very low CO 2 concentrations. 16 In recent studies, we found that both the formation of NHC-CO 2 adducts at 20-50°C in nearly 100% yield and their complete decomposition into free N-heterocyclic carbenes and CO 2 at elevated temperatures were very fast (Figure 1). 17 These observation stimulated us to explore the feasibility of N-heterocyclic carbenes as highly CO 2 -selective adsorbent at low CO 2 concentration. Herein, we report a N-heterocyclic carbene-functionalized synthetic polymer for reversible fixation-release of CO 2 .The synthetic route of zwitterionic NHC-CO 2 adduct-functionalized copolymer (designated as P-NHC-CO 2 ) is shown in Scheme 1. Formation of quaternary ammonium by reaction of 1-(2,6-diisopropylphenyl) imidazole with styrene-4-vinylbenzyl chloride copolymer afforded imidazolium-modified copolymer P-NHC-HCl. Similar to the synthesis of 1-(2,6-diisopropylphenyl)-3-benzyl imidazolium-2-carboxylate (see Supporting Information), P-NHC-CO 2 was readily prepared by deprotonation of P-NHC-HCl using KN(SiMe 3 ) 2 , and followed by reaction with CO 2 . The IR spectrum of the resulted polymer showed a broad absorption peak around 1649 cm -1 , which is assigned to the asymmetric v(CO 2 ) vibrations of NHC-CO 2 adduct moiety. The content of immobilized N-heterocyclic carbene sites could be c...