Drunken lipid membranes, not drunken SNARE proteins, promote fusion in a model of neurotransmitter release.

Alcohol affects many neuronal proteins that are upstream or down-stream of synaptic vesicle fusion and neurotransmitter release. Less well studied is alcohol's effect on the fusion machinery including SNARE proteins and lipid membranes. Using a SNARE-driven fusion assay we show that fusion probability is significantly increased at 0.4% v/v (68 mM) ethanol; but not with methanol up to 10%. Ethanol appears to act directly on membrane lipids since experiments focused on protein properties [circular dichroism spectrometry, site-directed fluorescence interference contrast (sdFLIC) microscopy, and vesicle docking results] showed no significant changes up to 5% ethanol, but a protein-free fusion assay also showed increased lipid membrane fusion rates with 0.4% ethanol. These data show that the effects of high physiological doses of ethanol on SNARE-driven fusion are mediated through ethanol's interaction with the lipid bilayer of membranes and not SNARE proteins, and that methanol affects lipid membranes and SNARE proteins only at high doses.

A review of TNP-ATP in protein binding studies: benefits and pitfalls.

We review 50 years of use of 2',3'-O-trinitrophenyl (TNP)-ATP, a fluorescently tagged ATP analog. It has been extensively used to detect binding interactions of ATP to proteins and to measure parameters of those interactions such as the dissociation constant, Kd, or inhibitor dissociation constant, Ki. TNP-ATP has also found use in other applications, for example, as a fluorescence marker in microscopy, as a FRET pair, or as an antagonist (e.g., of P2X receptors). However, its use in protein binding studies has limitations because the TNP moiety often enhances binding affinity, and the fluorescence changes that occur with binding can be masked or mimicked in unexpected ways. The goal of this review is to provide a clear perspective of the pros and cons of using TNP-ATP to allow for better experimental design and less ambiguous data in future experiments using TNP-ATP and other TNP nucleotides.

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers.

Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol's effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC50 = 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior.

Functional ryanodine receptors in the membranes of neurohypophysial secretory granules.

Highly localized Ca(2+) release events have been characterized in several neuronal preparations. In mouse neurohypophysial terminals (NHTs), such events, called Ca(2+) syntillas, appear to emanate from a ryanodine-sensitive intracellular Ca(2+) pool. Traditional sources of intracellular Ca(2+) appear to be lacking in NHTs. Thus, we have tested the hypothesis that large dense core vesicles (LDCVs), which contain a substantial amount of calcium, represent the source of these syntillas. Here, using fluorescence immunolabeling and immunogold-labeled electron micrographs of NHTs, we show that type 2 ryanodine receptors (RyRs) are localized specifically to LDCVs. Furthermore, a large conductance nonspecific cation channel, which was identified previously in the vesicle membrane and has biophysical properties similar to that of an RyR, is pharmacologically affected in a manner characteristic of an RyR: it is activated in the presence of the RyR agonist ryanodine (at low concentrations) and blocked by the RyR antagonist ruthenium red. Additionally, neuropeptide release experiments show that these same RyR agonists and antagonists modulate Ca(2+)-elicited neuropeptide release from permeabilized NHTs. Furthermore, amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Collectively, our findings suggest that RyR-dependent syntillas could represent mobilization of Ca(2+) from vesicular stores. Such localized vesicular Ca(2+) release events at the precise location of exocytosis could provide a Ca(2+) amplification mechanism capable of modulating neuropeptide release physiologically.

Resolving double disulfide bond patterns in SNAP25B using liquid chromatography-ion trap mass spectrometry.

Complex disulfide bond patterns in synaptosomal-associated protein of 25 kD B (SNAP25B) are thought to regulate neurotransmitter release in response to oxidative stress. However, the steric feasibility of each possible disulfide pattern in SNAP25B has not been assessed. To assess the steric feasibility of hypothesized closely spaced complex disulfide patterning in SNAP25B and also the feasibility of identifying complex disulfide bond patterns with MS, we have developed a novel probabilistic analysis to unambiguously resolve complex double disulfide bond patterns by using an ion trap mass spectrometer. We analyzed fragmentation patterns of singly linked peptides to determine likely fragmentation events in an ion trap mass spectrometer and observed double and single backbone cleavage along with heterolytic cleavage of the disulfide bond. We modeled these same events in the doubly disulfide linked SNAP25B peptide and used a cumulative hypergeometric distribution with top-down scoring to both identify and differentiate these bonding patterns. Because of the presence of unique MS/MS peaks, two of the bonding patterns were directly identified. The third was assigned on the basis of full chromatographic separation and confirmed by modeling triple breakage fragments. In total, this work demonstrates the feasibility--and also limitations--of identification of complex intradisulfide patterns by using ion trap-based collision-induced dissociation-based fragmentation methods.

Vesicle fusion to planar membranes is enhanced by cholesterol and low temperature.

Lipid composition and properties play an important role in many cellular properties such as fusion of vesicles to cell membranes, an essential process for exocytosis. Using a model system composed of artificial vesicles (liposomes) and artificial membranes (planar lipid bilayers), we observed that fusion is significantly affected by the lipid phase of the planar membrane. To determine the effect of lipid phases on fusion rates, we utilized the nystatin/ergosterol fusion assay and stimulated fusion with an osmotic gradient. Phase of the planar membrane was altered by changing cholesterol or temperature while the vesicular lipids were held constant. Liquid disordered (L(d) or L(α)) planar membranes were formed from phosphatidylethanolamine and phosphatidylcholine with unsaturated acyl chains. Addition of cholesterol shifts these membranes to the liquid ordered (L(o)) phase and increases liposome fusion. Planar membranes in the L(α) phase were also made from dipalmitoylphoshatidylcholine (DPPC) above the transition temperature (T(m)) of 41.5 ° C. Decreasing the temperature below T(m) shifts these membranes into the ripple phase (P(ß')) and also increases liposome fusion. The cholesterol and temperature data are consistent with the hypothesis that fusion is promoted in membranes that have greater exposure of their lipid tails or in membranes which can form leaflet domains with negative curvature. The data are not consistent with the hypothesis that lipid mismatch drives fusion.

An assay to quantitate reducible cysteines from nanograms of GST-fusion proteins.

Cysteine residues in proteins are targets of numerous post-translational modifications and play important roles in protein structure and enzymatic function. Consequently, understanding the full biochemistry of proteins depends on determining the oxidation state and availability of the residues to be modified. We have developed a highly sensitive assay that accurately determines the number of unmodified cysteine residues in GST-fusion proteins. Only picomoles of protein are required for each reaction, which are carried out in 96-well glutathione-coated plates. Free unmodified cysteine residues are labeled and quantified using biotin and HRP-conjugated streptavidin. Our assay can be used to quantify reactions targeting sulfhydryl groups in proteins. We demonstrate this assay using full-length and truncation mutants of the SNARE proteins syntaxin1A, SNAP-25B, and synaptobrevin2, which have 0-4 cysteines. We are able to accurately determine the number of cysteine residues in each protein and follow the modification of these cysteines by oxidation and reaction with NEM (N-ethylmaleimide). This assay is as simple as running an ELISA or Western blot and, because of its high resolution, should allow detailed analysis of the chemistry of cysteine residues in proteins.

Chemomechanical regulation of SNARE proteins studied with molecular dynamics simulations.

SNAP-25B is a neuronal protein required for neurotransmitter (NT) release and is the target of Botulinum Toxins A and E. It has two SNARE domains that form a four-helix bundle when combined with syntaxin 1A and synaptobrevin. Formation of the three-protein complex requires both SNARE domains of SNAP-25B to align parallel, stretching out a central linker. The N-terminal of the linker has four cysteines within eight amino acids. Palmitoylation of these cysteines helps target SNAP-25B to the membrane; however, these cysteines are also an obvious target for oxidation, which has been shown to decrease SNARE complex formation and NT secretion. Because the linker is only slightly longer than the SNARE complex, formation of a disulfide bond between two cysteines might shorten it sufficiently to reduce secretion by limiting complex formation. To test this idea, we have carried out molecular dynamics simulations of the SNARE complex in the oxidized and reduced states. Indeed, marked conformational differences and a reduction of helical content in SNAP-25B upon oxidation are seen. Further differences are found for hydrophobic interactions at three locations, crucial for the helix-helix association. Removal of the linker induced different conformational changes than oxidation. The simulations suggest that oxidation of the cysteines leads to a dysfunctional SNARE complex, thus downregulating NT release during oxidative stress.

Proton transport through influenza A virus M2 protein reconstituted in vesicles.

Influenza A virus M2 protein is known to form acid-activated, proton-selective, amantadine-sensitive channels. We directly measured proton uptake in vesicles containing reconstituted M2 by monitoring external pH after addition of valinomycin to vesicles with 100-fold-diluted external [K(+)]. External pH typically increased by a few tenths of a pH unit over a few minutes after valinomycin addition, but proton uptake was not significantly altered by acidification. Under neutral conditions, external addition of 1 mM amantadine produced a reduction in flux consistent with randomly ordered channels; however, experimental variation is high with this method and the block was not statistically significant. Amantadine block was reduced at pH 5.4. In accord with Lin and Schroeder's study of reconstituted M2 using a pH-sensitive dye to monitor intravesicular pH, we conclude that bath pH weakly affects or does not significantly affect proton flow in the pH range 5.4-7.0 for the reconstituted system, contrary to results from electrophysiological studies. Theoretical analysis of the relaxation to Donnan equilibrium utilized for such vesicle uptake assays illuminates the appropriate timescale of the initial slope and an important limitation that must be placed on inferences about channel ion selectivity. The rise in pH over 10 s after ionophore addition yielded time-averaged single-channel conductances of 0.35 +/- 0.20 aS and 0.72 +/- 0.42 aS at pH 5.4 and 7.0, respectively, an order of magnitude lower than previously reported in vesicles. Assuming complete membrane incorporation and tetramerization of the reconstituted protein, such a low time-averaged conductance in the face of previously observed single-channel conductance (6 pS at pH 3) implies an open channel probability of 10(-6)-10(-4). Based on leakage of potassium from M2-containing vesicles, compared to protein-free vesicles, we conclude that M2 exhibits approximately 10(7) selectivity for hydrogen over potassium.

The role of cavitation in liposome formation.

Liposome size is a vital parameter of many quantitative biophysical studies. Sonication, or exposure to ultrasound, is used widely to manufacture artificial liposomes, yet little is known about the mechanism by which liposomes are affected by ultrasound. Cavitation, or the oscillation of small gas bubbles in a pressure-varying field, has been shown to be responsible for many biophysical effects of ultrasound on cells. In this study, we correlate the presence and type of cavitation with a decrease in liposome size. Aqueous lipid suspensions surrounding a hydrophone were exposed to various intensities of ultrasound and hydrostatic pressures before measuring their size distribution with dynamic light scattering. As expected, increasing ultrasound intensity at atmospheric pressure decreased the average liposome diameter. The presence of collapse cavitation was manifested in the acoustic spectrum at high ultrasonic intensities. Increasing hydrostatic pressure was shown to inhibit the presence of collapse cavitation. Collapse cavitation, however, did not correlate with decreases in liposome size, as changes in size still occurred when collapse cavitation was inhibited either by lowering ultrasound intensity or by increasing static pressure. We propose a mechanism whereby stable cavitation, another type of cavitation present in sound fields, causes fluid shearing of liposomes and reduction of liposome size. A mathematical model was developed based on the Rayleigh-Plesset equation of bubble dynamics and principles of acoustic microstreaming to estimate the shear field magnitude around an oscillating bubble. This model predicts the ultrasound intensities and pressures needed to create shear fields sufficient to cause liposome size change, and correlates well with our experimental data.

Evidence that nystatin channels form at the boundaries, not the interiors of lipid domains.

Nystatin (nys) is an antifungal agent that preferentially forms ion channels in membranes containing the sterol, ergosterol (erg). The structure of the nystatin channel is not clear, but it is known that multiple nystatin monomers must aggregate to form channels in a sterol-rich membrane. When nys/erg containing vesicles are fused to a sterol-free bilayer, characteristic spikelike changes in membrane conductance are observed. An abrupt increase in conductance is followed by a decay that is generally stepwise linear and the decay time depends strongly on [erg]. These data are inconsistent with the hypothesis that nys channels form uniformly throughout the membrane and decay independently (which would produce exponential decay). We propose that channels are located at the boundaries of lipid superlattices such that diffusion of erg out of the lattice results in correlated channel decay. This was tested using a statistical mechanical analysis and Monte Carlo simulations, which reveal details of the diffusion process and provide insight into conditions at superlattice boundaries during decay. This analysis predicts the linear decay schemes and the dramatic drop in channel decay time observed at erg mol % = 50. This interpretation also explains puzzling data relating conductance spike height to vesicle diameter.

SNARE complex regulation by phosphorylation.

SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors) are ubiquitous proteins that direct vesicular trafficking and exocytosis. In neurons, SNAREs act to mediate release of neurotransmitters, which is a carefully regulated process. Calcium influx has long been shown to be the key trigger of release. However, calcium alone cannot regulate the degree of vesicle content release. For example, only a limited number of docked vesicles releases neurotransmitters when calcium entry occurs; this suggests that exocytosis is regulated by other factors besides calcium influx. Regulation of the degree of release is best explained by looking at the many enzymatic proteins that interact with the SNARE complex. These proteins have been hypothesized to regulate the formation, stability, or disassembly of the SNARE complex and therefore may regulate neurotransmitter release. One group of enzymatic regulators is the protein kinases. These proteins phosphorylate sites on both SNARE proteins and proteins that interact with SNARE proteins. Recent research has identified some of the specific effects that phosphorylation (or dephosphorylation) at these sites can produce. Additionally, palmitoylation of SNAP-25, regulates the localization, and hence activity of this key SNARE protein. This review focuses on the location and effects of phosphorylation on SNARE regulation.

Reducing liposome size with ultrasound: bimodal size distributions.

Sonication is a simple method for reducing the size of liposomes. We report the size distributions of liposomes as a function of sonication time using three different techniques. Liposomes, mildly sonicated for just 30 sec, had bimodal distributions when surface-weighted with modes at about 140 and 750 nm. With extended sonication, the size distribution remains bimodal but the average diameter of each population decreases and the smaller population becomes more numerous. Independent measurements of liposome size using Dynamic Light Scattering (DLS), transmission electron microscopy (TEM), and the nystatin/ergosterol fusion assay all gave consistent results. The bimodal distribution (even when number-weighted) differs from the Weibull distribution commonly observed for liposomes sonicated at high powers over long periods of time and suggests that a different mechanism may be involved in mild sonication. The observations are consistent with the following mechanism for decreasing liposome size. During ultrasonic irradiation, cavitation, caused by oscillating microbubbles, produces shear fields. Large liposomes that enter these fields form long tube-like appendages that can pinch-off into smaller liposomes. This proposed mechanism is consistent with colloidal theory and the observed behavior of liposomes in shear fields.

Syntaxin 1A drives fusion of large dense-core neurosecretory granules into a planar lipid bilayer.

The SNARE complex, involved in vesicular trafficking and exocytosis, is composed of proteins in the vesicular membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Our results show that modified large dense-core neurosecretory granules (NSGs), isolated from the bovine neurohypophysis, spontaneously fuse with a planar lipid membrane containing only the t-SNARE syntaxin 1A. This provides evidence that syntaxin alone is able to form a functional fusion complex with native v-SNAREs of the NSG. The fusion was similar to constitutive, not regulated, exocytosis because changes in free [Ca2+] had no effect on the syntaxin-mediated fusion. Several deletion mutants of syntaxin 1A were also tested. The removal of the regulatory domain did not significantly reduce spontaneous fusion. However, a syntaxin deletion mutant consisting of only the transmembrane domain was incapable of eliciting spontaneous fusion. Finally, a soluble form of syntaxin 1A (lacking its transmembrane domain) was used to saturate the free syntaxin-binding sites of modified NSGs. This treatment blocks spontaneous fusion of these granules to a bilayer containing full-length syntaxin 1A. This method provides an effective model system to study possible regulatory components affecting vesicle fusion.

Determination of proton flux and conductance at pH 6.8 through single FO sectors from Escherichia coli.

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H+) flux and conductance through a single FO of the F1FO ATP synthase. Lipid vesicles reconstituted with just a few functional FO from Escherichia coli were loaded with 250 mM K+ and suspended in a low K+ solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single FO sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type FO has a proton flux of 3100 +/- 500 H+/s/FO at a transmembrane potential of 106 mV (25 degrees C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.