Patch clamp studies of single cell-fusion events mediated by a viral fusion protein

To enter cells, viruses must fuse their envelope with a host cell membrane. Fusion is mediated by specific, membrane-spanning fusion proteins, of which the influenza virus haemagglutinins (HA) are the best characterized. Several HAs have been sequenced, and the crystal structure of the major part of one HA is known. The conditions for fusion and some of the rearrangements in the HA that accompany fusion are well understood, but it remains unclear how HA causes bilayers to fuse. We have observed, in real time, unitary cell-fusion events caused by HA. Fibroblasts expressing HA were induced to fuse with red blood cells by a rapid drop in pH. Fusion was monitored by fluorescence microscopy, and by measuring the membrane conductance and capacitance of the fibroblast. The earliest event observed was the sudden opening of an aqueous pore connecting the cytoplasms of the fusing cells. Initially, the pore conductance often fluctuated between zero and approximately 600 pS, as if the pore were opening and closing repeatedly. Later, it increased over tens of seconds, as if the pore dilated. We suggest that, as in exocytosis, HA-mediated membrane fusion begins with the formation of a narrow pore. Based on the conductance, we estimate the initial diameter of the pore to be no more than twice that of a gap junction channel.     

Vesicular restriction of synaptobrevin suggests a role for calcium in membrane fusion

Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis is triggered by calcium and requires three SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptors) proteins: synaptobrevin (also known as VAMP) on the synaptic vesicle, and syntaxin and SNAP-25 on the plasma membrane. Neuronal SNARE proteins form a parallel four-helix bundle that is thought to drive the fusion of opposing membranes. As formation of this SNARE complex in solution does not require calcium, it is not clear what function calcium has in triggering SNARE-mediated membrane fusion. We now demonstrate that whereas syntaxin and SNAP-25 in target membranes are freely available for SNARE complex formation, availability of synaptobrevin on synaptic vesicles is very limited. Calcium at micromolar concentrations triggers SNARE complex formation and fusion between synaptic vesicles and reconstituted target membranes. Although calcium does promote interaction of SNARE proteins between opposing membranes, it does not act by releasing synaptobrevin from synaptic vesicle restriction. Rather, our data suggest a mechanism in which calcium-triggered membrane apposition enables syntaxin and SNAP-25 to engage synaptobrevin, leading to membrane fusion.     

Quantification of Ca2+-activated K+ channels under hormonal control in pig pancreas acinar cells

Ca2+- and voltage-activated K+ channels are found in many electrically excitable cells and have an important role in regulating electrical activity. Recently, the large K+ channel has been found in the baso-lateral plasma membranes of salivary gland acinar cells, where it may be important in the regulation of salt transport. Using patch-clamp methods to record single-channel currents from excised fragments of baso-lateral acinar cell membranes in combination with current recordings from isolated single acinar cells and two- and three-cell clusters, we have now for the first time characterized the K+ channels quantitatively. In pig pancreatic acini there are 25-60 K+ channels per cell with a maximal single channel conductance of about 200 pS. We have quantified the relationship between internal ionized Ca2+ concentration [( Ca2+]i) membrane potential and open-state probability (p) of the K+ channel. By comparing curves obtained from excised patches relating membrane potential to p, at different levels of [Ca2+]i, with similar curves obtained from intact cells, [Ca2+]i in resting acinar cells was found to be between 10(-8) and 10(-7) M. In microelectrode experiments acetylcholine (ACh), gastrin-cholecystokinin (CCK) as well as bombesin peptides evoked Ca2+-dependent opening of the K+ conductance pathway, resulting in membrane hyperpolarization. The large K+ channel, which is under strict dual control by internal Ca2+ and voltage, may provide a crucial link between hormone-evoked increase in internal Ca2+ concentration and the resulting NaCl-rich fluid secretion.     

Two modes of fusion pore opening revealed by cell-attached recordings at a synapse

Fusion of a vesicle with the cell membrane opens a pore that releases transmitter to the extracellular space. The pore can either dilate fully so that the vesicle collapses completely, or close rapidly to generate 'kiss-and-run' fusion. The size of the pore determines the release rate. At synapses, the size of the fusion pore is unclear, 'kiss-and-run' remains controversial, and the ability of 'kiss-and-run' fusion to generate rapid synaptic currents is questionable. Here, by recording fusion pore kinetics during single vesicle fusion, we found both full collapse and 'kiss-and-run' fusion at calyx-type synapses. For full collapse, the initial fusion pore conductance (G(p)) was usually >375 pS and increased rapidly at > or =299 pS ms(-1). 'Kiss-and-run' fusion was seen as a brief capacitance flicker (<2 s) with G(p) >288 pS for most flickers, but within 15-288 pS for the remaining flickers. Large G(p) (>288 pS) might discharge transmitter rapidly and thereby cause rapid synaptic currents, whereas small G(p) might generate slow and small synaptic currents. These results show that 'kiss-and-run' fusion occurs at synapses and that it can generate rapid postsynaptic currents, and suggest that various fusion pore sizes help to control the kinetics and amplitude of synaptic currents.     

Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion

The question concerning whether all membranes fuse according to the same mechanism has yet to be answered satisfactorily. During fusion of model membranes or viruses, membranes dock, the outer membrane leaflets mix (termed hemifusion), and finally the fusion pore opens and the contents mix. Viral fusion proteins consist of a membrane-disturbing 'fusion peptide' and a helical bundle that pin the membranes together. Although SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes form helical bundles with similar topology, it is unknown whether SNARE-dependent fusion events on intracellular membranes proceed through a hemifusion state. Here we identify the first hemifusion state for SNARE-dependent fusion of native membranes, and place it into a sequence of molecular events: formation of helical bundles by SNAREs precedes hemifusion; further progression to pore opening requires additional peptides. Thus, SNARE-dependent fusion may proceed along the same pathway as viral fusion: both use a docking mechanism via helical bundles and additional peptides to destabilize the membrane and efficiently induce lipid mixing. Our results suggest that a common lipidic intermediate may underlie all fusion reactions of lipid bilayers.     

Endfeet of retinal glial cells have higher densities of ion channels that mediate K+ buffering

A major function of glial cells in the central nervous system is to buffer the extracellular potassium concentration, [K+]o. A local rise in [K+]o causes potassium ions to enter glial cells, which have membranes that are highly permeable to K+; potassium then leaves the glial cells at other locations where [K+]o has not risen. We report here the first study of the individual ion channels mediating potassium buffering by glial cells. The patch-clamp technique was employed to record single channel currents in Müller cells, the radial glia of the vertebrate retina. Those cells have 94% of their potassium conductance in an endfoot apposed to the vitreous humour, causing K+ released from active retinal neurones to be buffered preferentially to the vitreous. Recordings from patches of endfoot and cell body membrane show that a single type of inward-rectifying K+ channel mediates potassium buffering at both cell locations. The non-uniform density of K+ conductance is due to a non-uniform distribution of one type of K+ channel, rather than to the cell expressing high conductance channels at the endfoot and low conductance channels elsewhere on the cell.     

Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals

The vesicles that package neurotransmitters fall into two distinct classes, large dense-core vesicles (LDCVs) and small synaptic vesicles, the coexistence of which is widespread in nerve terminals. High resolution capacitance recording reveals unitary steps proportional to vesicle size. Measurements of capacitance steps during LDCV and secretory granule fusion in endocrine and immune cells have provided important insights into exocytosis; however, extending these measurements to small synaptic vesicles has proven difficult. Here we report single vesicle capacitance steps in posterior pituitary nerve terminals. These nerve terminals contain neuropeptide-laden LDCVs, as well as microvesicles. Microvesicles are similar to synaptic vesicles in size, morphology and molecular composition, but their contents are unknown. Capacitance steps of two characteristic sizes, corresponding with microvesicles and LDCVs, were detected in patches of nerve terminal membrane. Both types of vesicles fuse in response to depolarization-induced Ca(2+) entry. Both undergo a reversible fusion process commonly referred to as 'kiss-and-run', but only rarely. Fusion pores seen during microvesicle kiss-and-run have a conductance of 19 pS, 11 times smaller than LDCV fusion pores. Thus, LDCVs and microvesicles use structurally different intermediates during exocytosis.     

Voltage and Ca2+-activated K+ channel in baso-lateral acinar cell membranes of mammalian salivary glands

Nervous or hormonal stimulation of many exocrine glands evokes release of cellular K+ (ref. 1), as originally demonstrated in mammalian salivary glands2,3, and is associated with a marked increase in membrane conductance1,4,5. We now demonstrate directly, by using the patch-clamp technique6, the existence of a K+ channel with a large conductance localized in the baso-lateral plasma membranes of mouse and rat salivary gland acinar cells. The K+ channel has a conductance of approximately 250 pS in the presence of high K+ solutions on both sides of the membrane. Although mammalian exocrine glands are believed not to possess voltage-activated channels1,7, the probability of opening the salivary gland K+ channel was increased by membrane depolarization. The frequency of channel opening, particularly at higher membrane potentials, was increased markedly by elevating the internal ionized Ca2+ concentration, as previously shown for high-conductance K+ channels from cells of neural origin8-10. The Ca2+ and voltage-activated K+ channel explains the marked cellular K+ release that is characteristically observed when salivary glands are stimulated to secrete.     

Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2

Membrane transport between the endoplasmic reticulum and the plasma membrane, which involves the budding and fusion of carrier vesicles, is inhibited during mitosis in animal cells. At the same time, the Golgi complex and the nuclear envelope, as well as the endoplasmic reticulum in some cell types, become fragmented. Fragmentation of the Golgi is believed to facilitate its equal partitioning between daughter cells. In fact, it has been postulated that both the inhibition of membrane traffic and Golgi fragmentation during mitosis are due to an inhibition of vesicle fusion, while vesicle budding continues. Although less is known about the endocytic pathway, internalization and receptor recycling are also arrested during mitosis. We have now used a cell-free assay to show that the fusion of endocytic vesicles from baby hamster kidney cells is reduced in Xenopus mitotic cytosol when compared with interphase cytosol. We reconstituted this inhibition in interphase cytosol by adding a preparation enriched in the starfish homologue of the cdc2 protein kinase. Inhibition was greater than or equal to 90% when the added cdc2 activity was in the range estimated for that in mitotic Xenopus eggs, which indicates that during mitosis the cdc2 kinase mediates an inhibition of endocytic vesicle fusion, and possibly other fusion events in membrane traffic.     

Topological restriction of SNARE-dependent membrane fusion

To fuse transport vesicles with target membranes, proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex must be located on both the vesicle (v-SNARE) and the target membrane (t-SNARE). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 and Bet1 (refs 2-6), each probably contribute a single helix to form the SNARE complex that is needed for transport from endoplasmic reticulum to Golgi. This generates a four-helix bundle, which ultimately mediates the actual fusion event. Here we explore how the anchoring arrangement of the four helices affects their ability to mediate fusion. We reconstituted two populations of phospholipid bilayer vesicles, with the individual SNARE proteins distributed in all possible combinations between them. Of the eight non-redundant permutations of four subunits distributed over two vesicle populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restricted by design to function either as a v-SNARE or as part of a t-SNARE complex.     

Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans-complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2+/calmodulin-dependent fashion. V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion.     

Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum

Inositol 1,4,5-trisphosphate (InsP3) can initiate calcium release into the cytoplasm in a variety of cells. From experiments using permeabilized cells, membrane vesicles, and patch-clamp techniques, it has been suggested that InsP3 acts by directly opening calcium channels. Here, we show that InsP3 induced openings of channels in planar lipid bilayers into which vesicles made from aortic muscle sarcoplasmic reticulum (SR) were incorporated. Activation of channels by InsP3 was not observed when vesicles made from SR of cardiac or skeletal muscle were incorporated into planar lipid bilayers. The present study demonstrates for the first time unique properties of an InsP3-gated calcium channel in sarcoplasmic reticulum vesicles from vascular smooth muscle. This InsP3-activated channel from aortic SR differs strikingly from the calcium-gated calcium channel of striated muscle SR in single-channel conductance and pharmacology.     

Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes

The nicotinic acetylcholine receptor belongs to a class of molecules that respond transiently to chemical stimuli by opening a water-filled channel through the cell membrane for cations to diffuse. This channel lies along the central axis delineated by a ring of five homologous, membrane-spanning subunits and thus has properties, such as conductance and ion selectivity, which depend on the profile created by the encircling subunits. Insight has been gained recently about the amino-acid residues implicated directly in the ion transport, and some information about the subunit configuration around the channel has come from electron microscopy studies of postsynaptic membranes crystallized in the form of flattened tubular vesicles. The resolution along the axis of the channel has, however, been limited by the restricted range of views obtainable. Here we report the structure of the channel at 17 A resolution, determined by three-dimensional image reconstruction from tubular vesicles having receptors organized in helical arrays across their surfaces. The helical symmetry is preserved by suspending the tubes in thin films of ice, and the receptors in such tubes can be seen from all angles, allowing the channel to be revealed clearly in relation to the lipid bilayer and the peripheral protein for the first time.     

Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension

Lipid bilayer membranes--ubiquitous in biological systems and closely associated with cell function--exhibit rich shape-transition behaviour, including bud formation and vesicle fission. Membranes formed from multiple lipid components can laterally separate into coexisting liquid phases, or domains, with distinct compositions. This process, which may resemble raft formation in cell membranes, has been directly observed in giant unilamellar vesicles. Detailed theoretical frameworks link the elasticity of domains and their boundary properties to the shape adopted by membranes and the formation of particular domain patterns, but it has been difficult to experimentally probe and validate these theories. Here we show that high-resolution fluorescence imaging using two dyes preferentially labelling different fluid phases directly provides a correlation between domain composition and local membrane curvature. Using freely suspended membranes of giant unilamellar vesicles, we are able to optically resolve curvature and line tension interactions of circular, stripe and ring domains. We observe long-range domain ordering in the form of locally parallel stripes and hexagonal arrays of circular domains, curvature-dependent domain sorting, and membrane fission into separate vesicles at domain boundaries. By analysing our observations using available membrane theory, we are able to provide experimental estimates of boundary tension between fluid bilayer domains.     

Compartmental specificity of cellular membrane fusion encoded in SNARE proteins

Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.     

A role for ADP-ribosylation factor in nuclear vesicle dynamics.

Two distinct steps in nuclear envelope assembly can be assayed in vitro: the protein-mediated binding of nuclear-specific vesicles to chromatin, and the subsequent fusion of these vesicles to enclose the chromatin within a double nuclear membrane. Nuclear vesicle fusion, like fusion in the secretory pathway, requires ATP and cytosol and is inhibited by nonhydrolysable GTP analogues. The sensitivity of nuclear vesicle fusion to GTP-gamma S requires a GTP-dependent soluble factor, the properties of which are strikingly similar to a GTP-dependent Golgi binding factor (GGBF) that inhibits Golgi vesicle fusion in the presence of GTP-gamma S and belongs to the ADP-ribosylation factor (ARF) family of small GTPases. In the presence of GTP-gamma S, ARF proteins and alpha-, beta-, gamma-, delta-COP ('coatomer') subunits are associated with Golgi transport vesicles, but the exact roles of ARF proteins in secretion are not yet understood. We report here that purified ARF1 and GGBF have GTP-dependent soluble factor activity in the nuclear vesicle fusion assay. Our results show that the function of ARF is not limited to the Golgi apparatus, and indicate that there may be a link between the formation of nuclear vesicles during mitosis and proteins involved in secretion.     

Transmitter-like action of ATP on patched membranes of cultured myoblasts and myotubes

The concept of purinergic neurotransmission, first proposed by Burnstock, has been confirmed in various cell types. We show here, by the patch-clamp method, that external ATP in micromolar concentrations (1-100 microM) activates cation channels in the membranes of fusion-competent myoblasts and myotubes. In cell-attached membrane patches of myoblasts and myotubes the mean number of simultaneously activated channels increases with time after external ATP application. In myoblasts only one population of channels having a mean single-channel conductance of gamma=43 pS was found, while in myotubes two populations with gamma 1=48 pS and gamma 2=20 pS were observed. Treatment of myotube membranes with acetylcholine (ACh) or carbachol resulted in two populations of channels which had conductance values and voltage-dependent mean channel lifetimes similar to those produced in response to ATP. The results show that embryonic skeletal muscle cells contain cation channels sensitive to ATP and provide evidence for a neurotransmitter-like action of ATP on these cells.     

Single cyclic GMP-activated channel activity in excised patches of rod outer segment membrane

The plasma membrane of retinal rod outer segments contains a cyclic GMP-activated conductance which appears to be the light-sensitive conductance involved in phototransduction. Recently, it has been found that this conductance is partially blocked by Mg2+ and Ca2+ at physiological concentrations, thus possibly accounting for the absence of observable single-channel activity in excised membrane patches and for the unusually small apparent unit conductance deduced from noise measurements on intact cells. We now report that, as expected from this idea, single cGMP-activated channel activity can be detected from an excised rod membrane patch in the absence of divalent cations. The most prominent unitary current had a mean conductance of approximately 25 pS. Both individual channel openings (mean open time approximately 1 ms) and short bursts of openings (mean burst duration of about a few milliseconds) were observed. In addition, there were smaller events which probably represented other states of the conductance. The mean current increased with the third power of cGMP concentration, suggesting that there are at least three cGMP-binding sites on the channel molecule. With 0.2 mM Mg2+ in the cGMP-containing solution, a flickering block of the open channel was observed; the effect of Ca2+ was similar. The results resolve a puzzle about the light-sensitive conductance by demonstrating that it is an aqueous pore rather than a carrier.     

Single synaptic vesicles fusing transiently and successively without loss of identity

Vesicle fusion and recycling are particularly critical for ongoing neurotransmitter release in the small nerve terminals of the brain, which typically contain about 30 functional vesicles. However, the modes of exocytosis and endocytosis that operate at synapses of the central nervous system are incompletely understood. Here we show real-time visualization of a single vesicle fusing at a small synapse of the central nervous system, made possible by highly intensified charge-coupled device imaging of hippocampal synaptic terminals, in which a single vesicle was labelled with the fluorescent membrane marker FM1-43 (ref. 6). In a small number of cases, full loss of fluorescent membrane dye was elicited by a single action potential, consistent with classical complete collapse. In most cases, however, action potentials triggered only partial loss of fluorescence, suggesting vesicular retention of membrane marker, consistent with 'kiss-and-run' vesicle cycling. An alternative hypothesis of independent fusion of partially stained vesicles arising from endosomal splitting could be excluded by observations on the size and timing of successive fusion events. Thus, our experimental evidence supports a predominance of kiss-and-run fusion events and rapid vesicular re-use.     

Fast vesicle replenishment allows indefatigable signalling at the first auditory synapse

Ribbon-type synapses in inner hair cells of the mammalian cochlea encode the complexity of auditory signals by fast and tonic release through fusion of neurotransmitter-containing vesicles. At any instant, only about 100 vesicles are tethered to the synaptic ribbon, and about 14 of these are docked to the plasma membrane, constituting the readily releasable pool. Although this pool contains about the same number of vesicles as that of conventional synapses, ribbon release sites operate at rates of about two orders of magnitude higher and with submillisecond precision. How these sites replenish their vesicles so efficiently remains unclear. We show here, using two-photon imaging of single release sites in the intact cochlea, that preformed vesicles derived from cytoplasmic vesicle-generating compartments participate in fast release and replenishment. Vesicles were released at a maximal initial rate of 3 per millisecond during a depolarizing pulse, and were replenished at a rate of 1.9 per millisecond. We propose that such rapid resupply of vesicles enables temporally precise and sustained release rates. This may explain how the first auditory synapse can encode with indefatigable precision without having to rely on the slow, local endocytic vesicle cycle.