Jonathan R. Monck

THE EXOCYTOTIC FUSION PORE AND NEUROTRANSMITTER RELEASE

Membrane fusion is ubiquitous in biological systems, occurring in the simplest of unicellular eukaryotes as well as higher eukaryotes. As soon as the first primitive eukaryotic cell utilized a lipid bilayer as an outer membrane, membrane fusion (and fission) became necessary for the traffic of material from the outside to the inside, the inside to the outside, and between different intracellular membrane-bounded compartments. The earliest cells would have made use of the intrinsic ability of lipid bilayers to fuse under certain conditions. Although this fusogenic property of bilayers has been known for some time, it is has become clear only relatively recently that two phospholipid bilayers will fuse spontaneously, owing to a hydrophobic force, when the bilayers are brought close together under conditions of membrane tension or high curvature (Helm and Israelachvili, 1993). The primeval cell would have used proteins to develop the appropriate architecture in which such fusion would occur in a regulated manner. During the course of evolution, ever more sophisticated ways of regulating this basic process would evolve, but the underlying fusion mechanism would remain unchanged. We have proposed that a macromolecular scaffold of proteins is responsible for bringing the plasma membrane close to the secretory granule membranes and creating the architecture that enables the hydrophobic force to cause fusion (Figure 1; Nanavati et al., 1992; Monck and Fernandez, 1992; Oberhauser and Fernandez, 1993). Evidence is now accumulating that there are several highly conserved families of proteins associated with vesicle fusion events, from yeast to mammalian cells, and with intracellular traffic, as well as with regulated exocytosis and synaptic transmission (Bennett and Scheller, 1993; Sollner et al., 1993; Südhof et al., 1993). The molecular structures (or scaffolds) that regulate membrane fusion are likely to contain related proteins and share certain fundamental properties.

Pulsed laser imaging of rapid Ca2+ gradients in excitable cells

Excitable cells are thought to respond to action potentials by forming short lived and highly localized Ca2+ gradients near sites of Ca2+ entry or near the site of Ca2+ release by intracellular stores. However, conventional imaging techniques lack the spatial and temporal resolution to capture these gradients. Here we demonstrate the use of pulsed-laser microscopy to measure Ca2+ gradients with submicron spatial resolution and millisecond time resolution in two preparations where the Ca2+ signal is thought to be fast and highly localized: adrenal chromaffin cells, where the entry of Ca2+ through voltage dependent Ca2+ channels triggers exocytotic fusion; and skeletal muscle fibers, where intracellular Ca2+ release from the sarcoplasmic reticulum initiates contraction. In chromaffin cells, Ca2+ gradients developed over 10-100 ms and were initially restricted to discrete submembrane domains, or hot spots, before developing into complete rings of elevated Ca2+ concentration. In frog skeletal muscle large, short-lived (approximately 6 ms) Ca2+ gradients were observed within individual sarcomeres following induction of action potentials. The pulsed laser imaging approach permits, for the first time, the capture and critical examination of rapid Ca2+ signaling events such as those underlying excitation-secretion and excitation-contraction coupling.

Is swelling of the secretory granule matrix the force that dilates the exocytotic fusion pore

The swelling of the secretory granule matrix which follows fusion has been proposed as the driving force for the rapid expansion of the fusion pore necessary for exocytosis. To test this hypothesis, we have combined simultaneous measurements of secretory granule swelling using videomicroscopy with patch clamp measurements of the time course of the exocytotic fusion pore in mast cells from the beige mouse. We show that isotonic acidic histamine solutions are able to inhibit swelling of the secretory granule matrix both in purified secretory granules lysed by electroporation and in intact cells stimulated to exocytose by guanine nucleotides. In contrast to the inhibitory effects on granule swelling, the rate of expansion of the exocytotic fusion pore is unaffected. Therefore, as the rate of granule swelling was more than 20 times slower under these conditions, swelling of the secretory granule matrix due to water entry through the fusion pore cannot be the force responsible for the characteristic rapid expansion of the exocytotic fusion pore. We suggest that tension in the secretory granule membrane, which has recently been demonstrated in fused secretory granules, might be the force that drives the irreversible expansion of the fusion pore.

Events leading to the opening and closing of the exocytotic fusion pore have markedly different temperature dependencies. Kinetic analysis of single fusion events in patch-clamped mouse mast cells

The earliest event in exocytosis is the formation of a fusion pore, an aqueous channel that connects the lumen of a secretory granule with the extracellular space. We can observe the formation of individual fusion pores and their subsequent dilation or closure by measuring the changes in the admittance of patch-clamped mast cells during GTP gamma S-stimulated exocytotic fusion. To investigate the molecular structure of the fusion pore, we have studied the temperature dependency of the rate constants for fusion pore formation and closure. An Arrhenius plot of the rate of fusion pore formation shows a simple linear relationship with an apparent activation energy of 23 kcal/mol. In contrast, the Arrhenius plot of the rate of closure of the fusion pore is discontinuous, with the break at approximately 13 degrees C. Above the break point, the rate of closure has a weak temperature dependence (7 kcal/mol), whereas below 13 degrees C the rate of closure is temperature independent. This type of temperature dependency is characteristic of events that depend on diffusion in a lipid phase that undergoes a fluid-solid phase transition. We propose that the formation of the fusion pore is regulated by the conformational change of a molecular structure with a high activation energy, whereas the closure of the fusion pore is regulated by lipids that become phase separated at 13 degrees C.

The exocytotic fusion pore interface: a model of the site of neurotransmitter release.

Ultrastructural techniques have shown that an early event in the exocytotic fusion of a secretory vesicle is the formation of a narrow, water-filled pore spanning both the vesicle and plasma membranes and connecting the lumen of the secretory vesicle to the extracellular environment. Smaller precursors of the exocytotic fusion pore have been detected using electrophysiological techniques, which reveal a dynamic fusion pore that quickly expands to the size of the pores seen with electron microscopy. While it is clear that in the latter stages of expansion, when the size of the fusion pore is several orders of magnitude bigger than any known macromolecule, the fusion pore must be mainly made of lipids, the structure of the smaller precursors is unknown. Patch-clamp measurements of the activity of individual fusion pores in mast cells have shown that the fusion pore has some unusual and unexpected properties, namely that there is a large flux of lipid through the pore and the rate of pore closure has a discontinuous temperature dependency, suggesting a purely lipidic fusion pore. Moreover, comparisons of experimental data with theoretical fusion pores and with breakdown pores support the view that the fusion pore is initially a pore through a single bilayer, as would be expected for membrane fusion proceeding through a hemifusion mechanism. Based on these observations we present a model where the fusion pore is initially a pore through a single bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

GTPγS stimulates exocytosis in patch-clamped rat melanotrophs

We investigated the molecular mechanisms regulating exocytosis in patch-clamped melanotrophs by measuring the membrane capacitance. Ca(2+)-dependent exocytosis could be induced by membrane depolarization or by including solutions containing 2 microM free Ca2+ in the patch pipette. Ca(2+)-dependent exocytosis was inhibited by GDP beta S, suggesting involvement of a GTP-binding protein. The hydrolysis-resistant GTP analogs, GTP gamma S and GppNHp, were able to stimulate exocytosis at low free Ca2+ concentrations. The stimulatory response to GTP gamma S was abolished by both GDP beta S and GTP. The latter suggests that a sustained activation of a GTP-binding protein is necessary for exocytosis. This behavior is similar to the stimulation of exocytosis by guanine nucleotides in mast cells and other nonexcitable cells and suggests a common regulatory mechanism.