A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.

The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.

Tethering guides fusion-competent trans-SNARE assembly.

R-SNAREs (soluble N-ethylmaleimide-sensitive factor receptor), Q-SNAREs, and Sec1/Munc18 (SM)-family proteins are essential for membrane fusion in exocytic and endocytic trafficking. The yeast vacuolar tethering/SM complex HOPS (homotypic fusion and vacuole protein sorting) increases the fusion of membranes bearing R-SNARE to those with 3Q-SNAREs far more than it enhances their trans-SNARE pairings. We now report that the fusion of these proteoliposomes is also supported by GST-PX or GST-FYVE, recombinant dimeric proteins which tether by binding the phosphoinositides in both membranes. GST-PX is purely a tether, as it supports fusion without SNARE recognition. GST-PX tethering supports the assembly of new, active SNARE complexes rather than enhancing the function of the fusion-inactive SNARE complexes which had spontaneously formed in the absence of a tether. When SNAREs are more disassembled, as by Sec17, Sec18, and ATP (adenosine triphosphate), HOPS is required, and GST-PX does not suffice. We propose a working model where tethering orients SNARE domains for parallel, active assembly.

Mechanistic insights into the SNARE complex disassembly.

NSF (N-ethylmaleimide-sensitive factor) and α-SNAP (α-soluble NSF attachment protein) bind to the SNARE (soluble NSF attachment protein receptor) complex, the minimum machinery to mediate membrane fusion, to form a 20S complex, which disassembles the SNARE complex for reuse. We report the cryo-EM structures of the α-SNAP-SNARE subcomplex and the NSF-D1D2 domain in the 20S complex at 3.9- and 3.7-Å resolutions, respectively. Combined with the biochemical and electrophysiological analyses, we find that α-SNAPs use R116 through electrostatic interactions and L197 through hydrophobic interactions to apply force mainly on two positions of the VAMP protein to execute disassembly process. Furthermore, we define the interaction between the amino terminus of the SNARE helical bundle and the pore loop of the NSF-D1 domain and demonstrate its essential role as a potential anchor for SNARE complex disassembly. Our studies provide a rotation model of α-SNAP-mediated disassembly of the SNARE complex.

Single-Molecule Optical Tweezers Study of Regulated SNARE Assembly.

Intracellular membrane fusion mediates material and information exchange among different cells or cellular compartments with high accuracy and spatiotemporal resolution. Fusion is driven by ordered folding and assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) and regulated by many other proteins. Understanding regulated SNARE assembly is key to dissecting mechanisms and physiologies of various fusion processes and their associated diseases. Yet, it remains challenging to study regulated SNARE assembly using traditional ensemble-based experimental approaches. Here, we describe our new method to measure the energy and kinetics of neuronal SNARE assembly in the presence of α-SNAP, using a single-molecule manipulation approach based on high-resolution optical tweezers. Detailed experimental protocols and methods of data analysis are shown. This approach can be widely applied to elucidate the effects of regulatory proteins on SNARE assembly and membrane fusion.

Use of Surface Plasmon Resonance (SPR) to Determine Binding Affinities and Kinetic Parameters Between Components Important in Fusion Machinery.

Surface plasmon resonance (SPR) can be used to analyze both binding affinities and kinetic parameters between a ligand and an analyte. SPR can be performed by either cross-linking a given ligand to a sensor chip covalently or utilizing high-affinity non-covalent interactions to secure a ligand in a particular conformation to a chip, both of which have their potential advantages. SPR measurements are mass based and reflect the proportional amount of analyte bound to a given ligand at a given concentration when flowed at a set rate in order to determine the binding parameters of a given biochemical interaction. The resultant sensorgram can indicate different types of binding events as well as provide both ka and kd, which can be used to determine an equilibrium dissociation constant KD. SPR can be used to measure binding affinity of proteins involved in fusion such as between SNAREs, SNAREs, and proteins that interact with them such as Sec18 (NSF) or Sec17 (alpha-SNAP), or to measure the binding of any fusion-related protein to a specific lipid or other small molecules; however, KDs are determined by SPR using a titration of concentrations of analyte and a maximum point on the sensorgram signifying saturation of the protein in order to determine a steady-state KD.

Multiple paralogues of α-SNAP in Giardia lamblia exhibit independent subcellular localization and redistribution during encystation and stress.

BACKGROUND: The differently-diverged parasitic protist Giardia lamblia is known to have minimal machinery for vesicular transport. Yet, it has three paralogues of SNAP, a crucial component that together with NSF brings about disassembly of the cis-SNARE complex formed following vesicle fusion to target membranes. Given that most opisthokont hosts of this gut parasite express only one α-SNAP, this study was undertaken to determine whether these giardial SNAP proteins have undergone functional divergence. RESULTS: All three SNAP paralogues are expressed in trophozoites, encysting trophozoites and cysts. Even though one of them clusters with γ-SNAP sequences in a phylogenetic tree, functional complementation analysis in yeast indicates that all the three proteins are functionally orthologous to α-SNAP. Localization studies showed a mostly non-overlapping distribution of these α-SNAPs in trophozoites, encysting cells and cysts. In addition, two of the paralogues exhibit substantial subcellular redistribution during encystation, which was also seen following exposure to oxidative stress. However, the expression of the three genes remained unchanged during this redistribution process. There is also a difference in the affinity of each of these α-SNAP paralogues for GlNSF. CONCLUSIONS: None of the genes encoding the three α-SNAPs are pseudogenes and the encoded proteins are likely to discharge non-redundant functions in the different morphological states of G. lamblia. Based on the difference in the interaction of individual α-SNAPs with GlNSF and their non-overlapping pattern of subcellular redistribution during encystation and under stress conditions, it may be concluded that the three giardial α-SNAP paralogues have undergone functional divergence. Presence of one of the giardial α-SNAPs at the PDRs of flagella, where neither GlNSF nor any of the SNAREs localize, indicates that this α-SNAP discharges a SNARE-independent role in this gut pathogen.

Structural principles of SNARE complex recognition by the AAA+ protein NSF.

The recycling of SNARE proteins following complex formation and membrane fusion is an essential process in eukaryotic trafficking. A highly conserved AAA+ protein, NSF (N-ethylmaleimide sensitive factor) and an adaptor protein, SNAP (soluble NSF attachment protein), disassemble the SNARE complex. We report electron-cryomicroscopy structures of the complex of NSF, αSNAP, and the full-length soluble neuronal SNARE complex (composed of syntaxin-1A, synaptobrevin-2, SNAP-25A) in the presence of ATP under non-hydrolyzing conditions at ~3.9 Å resolution. These structures reveal electrostatic interactions by which two αSNAP molecules interface with a specific surface of the SNARE complex. This interaction positions the SNAREs such that the 15 N-terminal residues of SNAP-25A are loaded into the D1 ring pore of NSF via a spiral pattern of interactions between a conserved tyrosine NSF residue and SNAP-25A backbone atoms. This loading process likely precedes ATP hydrolysis. Subsequent ATP hydrolysis then drives complete disassembly.

Sec17/Sec18 act twice, enhancing membrane fusion and then disassembling cis-SNARE complexes.

At physiological protein levels, the slow HOPS- and SNARE-dependent fusion which occurs upon complete SNARE zippering is stimulated by Sec17 and Sec18:ATP without requiring ATP hydrolysis. To stimulate, Sec17 needs its central residues which bind the 0-layer of the SNARE complex and its N-terminal apolar loop. Adding a transmembrane anchor to the N-terminus of Sec17 bypasses this requirement for apolarity of the Sec17 loop, suggesting that the loop functions for membrane binding rather than to trigger bilayer rearrangement. In contrast, when complete C-terminal SNARE zippering is prevented, fusion strictly requires Sec18 and Sec17, and the Sec17 apolar loop has functions beyond membrane anchoring. Thus Sec17 and Sec18 act twice in the fusion cycle, binding to trans-SNARE complexes to accelerate fusion, then hydrolyzing ATP to disassemble cis-SNARE complexes.

Characterization of the Soluble NSF Attachment Protein gene family identifies two members involved in additive resistance to a plant pathogen.

Proteins with Tetratricopeptide-repeat (TPR) domains are encoded by large gene families and distributed in all plant lineages. In this study, the Soluble NSF-Attachment Protein (SNAP) subfamily of TPR containing proteins is characterized. In soybean, five members constitute the SNAP gene family: GmSNAP18, GmSNAP11, GmSNAP14, GmSNAP02, and GmSNAP09. Recently, GmSNAP18 has been reported to mediate resistance to soybean cyst nematode (SCN). Using a population of recombinant inbred lines from resistant and susceptible parents, the divergence of the SNAP gene family is analysed over time. Phylogenetic analysis of SNAP genes from 22 diverse plant species showed that SNAPs were distributed in six monophyletic clades corresponding to the major plant lineages. Conservation of the four TPR motifs in all species, including ancestral lineages, supports the hypothesis that SNAPs were duplicated and derived from a common ancestor and unique gene still present in chlorophytic algae. Syntenic analysis of regions harbouring GmSNAP genes in soybean reveals that this family expanded from segmental and tandem duplications following a tetraploidization event. qRT-PCR analysis of GmSNAPs indicates a co-regulation following SCN infection. Finally, genetic analysis demonstrates that GmSNAP11 contributes to an additive resistance to SCN. Thus, GmSNAP11 is identified as a novel minor gene conferring resistance to SCN.

Cryo-EM structure of SNAP-SNARE assembly in 20S particle.

N-ethylmaleimide-sensitive factor (NSF) and α soluble NSF attachment proteins (α-SNAPs) work together within a 20S particle to disassemble and recycle the SNAP receptor (SNARE) complex after intracellular membrane fusion. To understand the disassembly mechanism of the SNARE complex by NSF and α-SNAP, we performed single-particle cryo-electron microscopy analysis of 20S particles and determined the structure of the α-SNAP-SNARE assembly portion at a resolution of 7.35 Å. The structure illustrates that four α-SNAPs wrap around the single left-handed SNARE helical bundle as a right-handed cylindrical assembly within a 20S particle. A conserved hydrophobic patch connecting helices 9 and 10 of each α-SNAP forms a chock protruding into the groove of the SNARE four-helix bundle. Biochemical studies proved that this structural element was critical for SNARE complex disassembly. Our study suggests how four α-SNAPs may coordinate with the NSF to tear the SNARE complex into individual proteins.

Mechanistic insights into the recycling machine of the SNARE complex.

Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.

Three αSNAP and 10 ATP molecules are used in SNARE complex disassembly by N-ethylmaleimide-sensitive factor (NSF).

The fusion of intracellular membranes is driven by the formation of a highly stable four-helix bundle of SNARE proteins embedded in the vesicle and target membranes. N-Ethylmaleimide sensitive factor recycles SNAREs after fusion by binding to the SNARE complex through an adaptor protein, αSNAP, and using the energy of ATP hydrolysis to disassemble the complex. Although only a single molecule of αSNAP binds to a soluble form of the SNARE complex, we find that three molecules of αSNAP are used for SNARE complex disassembly. We describe an engineered αSNAP trimer that supports more efficient SNARE complex disassembly than monomeric αSNAP. Using the trimerized αSNAP, we find that N-ethylmaleimide-sensitive factor hydrolyzes 10 ATP molecules on average to disassemble a single SNARE complex.

G protein-dependent basal and evoked endothelial cell vWF secretion.

von Willebrand factor (vWF) secretion by endothelial cells (ECs) is essential for hemostasis and thrombosis; however, the molecular mechanisms are poorly understood. Interestingly, we observed increased bleeding in EC-Gα13(-/-);Gα12(-/-) mice that could be normalized by infusion of human vWF. Blood from Gα12(-/-) mice exhibited significantly reduced vWF levels but normal vWF multimers and impaired laser-induced thrombus formation, indicating that Gα12 plays a prominent role in EC vWF secretion required for hemostasis and thrombosis. In isolated buffer-perfused mouse lungs, basal vWF levels were significantly reduced in Gα12(-/-), whereas thrombin-induced vWF secretion was defective in both EC-Gαq(-/-);Gα11(-/-) and Gα12(-/-) mice. Using siRNA in cultured human umbilical vein ECs and human pulmonary artery ECs, depletion of Gα12 and soluble N-ethylmaleimide-sensitive-fusion factor attachment protein α (α-SNAP), but not Gα13, inhibited both basal and thrombin-induced vWF secretion, whereas overexpression of activated Gα12 promoted vWF secretion. In Gαq, p115 RhoGEF, and RhoA-depleted human umbilical vein ECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal secretion was unchanged. Finally, in vitro binding assays revealed that Gα12 N-terminal residues 10-15 mediated the binding of Gα12 to α-SNAP, and an engineered α-SNAP binding-domain minigene peptide blocked basal and evoked vWF secretion. Discovery of obligatory and complementary roles of Gα12 and Gαq/11 in basal vs evoked EC vWF secretion may provide promising new therapeutic strategies for treatment of thrombotic disease.

Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between α-soluble NSF attachment protein (SNAP) and SNARE complex.

Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

BKV agnoprotein interacts with α-soluble N-ethylmaleimide-sensitive fusion attachment protein, and negatively influences transport of VSVG-EGFP.

BACKGROUND: The human polyomavirus BK (BKV) infects humans worldwide and establishes a persistent infection in the kidney. The BK virus genome encodes three regulatory proteins, large and small tumor-antigen and the agnoprotein, as well as the capsid proteins VP1 to VP3. Agnoprotein is conserved among BKV, JC virus (JCV) and SV40, and agnoprotein-deficient mutants reveal reduced viral propagation. Studies with JCV and SV40 indicate that their agnoproteins may be involved in transcription, replication and/or nuclear and cellular release of the virus. However, the exact function(s) of agnoprotein of BK virus remains elusive. PRINCIPAL FINDINGS: As a strategy of exploring the functions of BKV agnoprotein, we decided to look for cellular interaction partners for the viral protein. Several partners were identified by yeast two-hybrid assay, among them α-SNAP which is involved in disassembly of vesicles during secretion. BKV agnoprotein and α-SNAP were found to partially co-localize in cells, and a complex consisting of agnoprotein and α-SNAP could be co-immunoprecipitated from cells ectopically expressing the proteins as well as from BKV-transfected cells. The N-terminal part of the agnoprotein was sufficient for the interaction with α-SNAP. Finally, we could show that BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter suggesting that agnoprotein may modulate exocytosis. CONCLUSIONS: We have identified the first cellular interaction partner for BKV agnoprotein. The most N-terminal part of BKV agnoprotein is involved in the interaction with α-SNAP. Presence of BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter.

Synaptotagmin 1 modulates lipid acyl chain order in lipid bilayers by demixing phosphatidylserine.

Synaptotagmin 1 (syt1) functions as the Ca(2+) sensor in neuronal exocytosis, and it has been proposed to act by modulating lipid bilayer curvature. Here we examine the effect of the two C2 domains (C2A and C2B) of syt1 on membrane lipid order and lateral organization. In mixtures of phosphatidylcholine and phosphatidylserine (PS), attenuated total internal reflection Fourier transform infrared spectroscopy indicates that a fragment containing both domains (C2AB) or C2B alone disorders the lipid acyl chains, whereas the C2A domain has little effect upon chain order. Two observations suggest that these changes reflect a demixing of PS. First, the changes in acyl chain order are reversed at higher protein concentration; second, selective lipid deuteration demonstrates that the changes in lipid order are associated only with the PS component of the bilayer. Independent evidence for lipid demixing is obtained from fluorescence self-quenching of labeled lipid and from natural abundance (13)C NMR, where heteronuclear single quantum correlation spectra reveal Ca(2+)-dependent chemical shift changes for PS, but not for phosphatidylcholine, in the presence of the syt1 C2 domains. The ability of syt1 to demix PS is observed in a range of lipid mixtures that includes cholesterol, phosphatidylethanolamine, and varied PS content. These data suggest that syt1 might facilitate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors)-mediated membrane fusion by phase separating PS, a process that is expected to locally buckle bilayers and disorder lipids due to the curvature tendencies of PS.

Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones.

The homotypic fusion of yeast vacuoles, each with 3Q- and 1R-SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q-SNAREs on one and the R-SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion-competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.

Structure and dynamics of gamma-SNAP: insight into flexibility of proteins from the SNAP family.

Soluble N-ethylmaleimide-sensitive factor attachment protein gamma (gamma-SNAP) is a member of an eukaryotic protein family involved in intracellular membrane trafficking. The X-ray structure of Brachydanio rerio gamma-SNAP was determined to 2.6 A and revealed an all-helical protein comprised of an extended twisted-sheet of helical hairpins with a helical-bundle domain on its carboxy-terminal end. Structural and conformational differences between multiple observed gamma-SNAP molecules and Sec17, a SNAP family protein from yeast, are analyzed. Conformational variation in gamma-SNAP molecules is matched with great precision by the two lowest frequency normal modes of the structure. Comparison of the lowest-frequency modes from gamma-SNAP and Sec17 indicated that the structures share preferred directions of flexibility, corresponding to bending and twisting of the twisted sheet motif. We discuss possible consequences related to the flexibility of the SNAP proteins for the mechanism of the 20S complex disassembly during the SNAP receptors recycling.

Conformational changes in the AAA ATPase p97-p47 adaptor complex.

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.

Trafficking of presynaptic AMPA receptors mediating neurotransmitter release: neuronal selectivity and relationships with sensitivity to cyclothiazide.

Postsynaptic glutamate AMPA receptors (AMPARs) can recycle between plasma membrane and intracellular pools. In contrast, trafficking of presynaptic AMPARs has not been investigated. AMPAR surface expression involves interactions between the GluR2 carboxy tail and various proteins including glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), protein interacting with C kinase 1 (PICK1), N-ethyl-maleimide-sensitive fusion protein (NSF). Here, peptides known to selectively block the above interactions were entrapped into synaptosomes to study the effects on the AMPA-evoked release of [3H]noradrenaline ([3H]NA) and [3H]acetylcholine ([3H]ACh) from rat hippocampal and cortical synaptosomes, respectively. Internalization of pep2-SVKI to prevent GluR2-GRIP/ABP/PICK1 interactions potentiated the AMPA-evoked release of [3H]NA but left unmodified that of [3H]ACh. Similar potentiation was caused by pep2-AVKI, the blocker of GluR2-PICK1 interaction. Conversely, a decrease in the AMPA-evoked release of [3H]NA, but not of [3H]ACh, was caused by pep2m, a selective blocker of the GluR2-NSF interaction. In the presence of pep2-SVKI the presynaptic AMPARs on noradrenergic terminals lost sensitivity to cyclothiazide. AMPARs releasing [3H]ACh, but not those releasing [3H]NA, were sensitive to spermine, suggesting that they are GluR2-lacking AMPARs. To conclude: (i) release-regulating presynaptic AMPARs constitutively cycle in isolated nerve terminals; (ii) the process exhibits neuronal selectivity; (iii) AMPAR trafficking and desensitization may be interrelated.