Relationship between fusion pore opening and release during mast cell exocytosis studied with patch amperometry.

We have studied exocytosis in rat peritoneal mast cells by cell-attached patch amperometry. Step increases in capacitance were accompanied by typical amperometric spikes due to the release of 5-hydroxytryptamine (serotonin), indicating exocytosis of typical mast cell granules. We have measured the time course of fusion pore expansion, and correlated it with release from the granule matrix. The fusion pore of mast cell granules grows in three stages. The initial expansion of the pore occurred at a rate of 5 nS/s, and in many cases an observable amperometric foot was detected. A second, rapid expansion phase occurred with a rate as high as 1000 nS/s, coinciding with the upstroke of the amperometric spike. A third, slower phase, with a rate of 5 nS/s, completed the final expansion of the fusion pore. These data reveal the very late stages in the exocytotic process, and demonstrate that the size of the fusion pore does not limit release during the upstroke of the amperometric spike or during the final, slow expansion that occurs during for the decay of the amperometric spike.

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells.

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the 'kiss-and-run' mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.

Analysis of SCAMP1 function in secretory vesicle exocytosis by means of gene targeting in mice.

Secretory carrier membrane proteins (SCAMPs) comprise a family of ubiquitous membrane proteins of transport vesicles with no known function. Their universal presence in all cells suggests a fundamental role in membrane traffic. SCAMPs are particularly highly expressed in organelles that undergo regulated exocytosis, such as synaptic vesicles and mast cell granules. Of the three currently known SCAMPs, SCAMP1 is the most abundant. To investigate the possible functions of SCAMP1, we generated mice that lack SCAMP1. SCAMP1-deficient mice are viable and fertile. They exhibit no changes in the overall architecture or the protein composition of the brain or alterations in peripheral organs. Capacitance measurements in mast cells demonstrated that exocytosis could be triggered reliably by GTPgammaS in SCAMP1-deficient cells. The initial overall capacitance of mast cells was similar between wild type and mutant mice, but the final cell capacitance after completion of exocytosis, was significantly smaller in SCAMP1-deficient cells than in wild type cells. Furthermore, there was an increased proportion of reversible fusion events, which may have caused the decrease in the overall capacitance change observed after exocytosis. Our data show that SCAMP1 is not essential for exocytosis, as such, and does not determine the stability or size of secretory vesicles, but is required for the full execution of stable exocytosis in mast cells. This phenotype could be the result of a function of SCAMP1 in the formation of stable fusion pores during exocytosis or of a role of SCAMP1 in the regulation of endocytosis after formation of fusion pores.

[Neurotransmitter release: a process of membrane fusion occurring in fractions of milliseconds]. / Liberación de neurotransmisores: un proceso de fusión de membranas que transcurre en fracciones de milisegundos.

INTRODUCTION: The physiological mechanisms involved in neurotransmitter release were established thanks to the pioneer work of Katz, Del Castillo and Miledi at the neuromuscular junction. They termed their work as the quantal hypothesis of synaptic transmission. This hypothesis was further established morphologically by the work of Heuser and Reese. However, the molecular events underlying this process are poorly understood. We are starting to know which proteins interact between the vesicle and plasma membrane to promote fusion and to identify which molecules participate in the sensing of cytosolic calcium. DEVELOPMENT: Thanks to the combination of molecular biology, electrophysiology and microfluorometry, a huge amount of new information is been obtained on the mechanisms participating in synaptic transmission. This data deals with processes concerning to different pools of synaptic vesicles and their availability to reach the presynaptic membrane; the molecular events responsible for the targeting of these vesicles with the plasma membrane; the sensitivity to calcium at the presynaptic membrane; the fusion of membranes required to release the vesicle contents, and the mechanisms responsible for membrane retrieval needed for presynaptic homeostasis. CONCLUSIONS: In this review we discuss new data regarding synaptic function. However, some key points are still a matter of controversy. Meanwhile the quantal hypothesis is valid, the precise processes by which channels and vesicles interact, membrane is recycled and vesicles reused are still controversial. New techniques should be developed to address these points.

The exocytotic event in chromaffin cells revealed by patch amperometry.

In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at <3 nm for a variable period, which can last for several seconds, before it expands. Transmitter is released much faster through this pore than in mast cells, generating a 'foot' signals which precedes the amperometric spike. Occasionally, the narrow pore forms only transiently and does not expand, allowing complete transmitter release without full fusion of the vesicle with the plasma membrane.

Oxygen sensing by ion channels and chemotransduction in single glomus cells.

We have monitored cytosolic [Ca2+] and dopamine release in intact fura-2-loaded glomus cells with microfluoroimetry and a polarized carbon fiber electrode. Exposure to low PO2 produced a rise of cytosolic [Ca2+] with two distinguishable phases: an initial period (with PO2 values between 150 and approximately 70 mm Hg) during which the increase of [Ca2+] is very small and never exceeds 150-200 nM, and a second phase (with PO2 below approximately 70 mm Hg) characterized by a sharp rise of cytosolic [Ca2+]. Secretion occurs once cytosolic [Ca2+] reaches a threshold value of 180 +/- 43 nM. The results demonstrate a characteristic relationship between PO2 and transmitter secretion at the cellular level that is comparable with the relation described for the input (O2 tension)output (afferent neural discharges) variables in the carotid body. Thus, the properties of single glomus cells can explain the sensory functions of the entire organ. In whole-cell, patch-clamped cells, we have found that in addition to O2-sensitive K+ channels, there are Ca2+ channels whose activity is also regulated by PO2. Ca2+ channel activity is inhibited by hpoxia, although in a strongly voltage-dependent manner. The average hypoxic inhibition of the calcium current in 30% +/- 10% at -20 mV but only 2% +/- 2% at +30 mV. The differential inhibition of K+ and Ca2+ channels by hypoxia helps to explain why the secretory response of the cells is displaced toward PO2 values (below approximately 70 mm Hg) within the range of those normally existing in arterial blood. These data provide a conceptual framework for understanding the cellular mechanisms of O2 chemotransduction in the carotid body.

Cytosolic calcium facilitates release of secretory products after exocytotic vesicle fusion.

We monitored single vesicle exocytosis by simultaneous measurements of cell membrane capacitance as an indicator of fusion and amperometric detection of serotonin release. We show here that vesicle-plasma membrane fusion in rat mast cell granules is followed by a variable, exponentially distributed, delay before bulk release. This delay reflects the time required for the expansion of the exocytotic fusion pore, lasting, on average, 231 ms in resting cytosolic calcium, [Ca2+]i (50 nM). In the presence of [Ca2+]i in the low micromollar range, the lag between fusion and release was reduced to 123 ms. The characteristics of the amperometric signals were unchanged by [Ca2+]i. These results show a novel site of regulation in the exocytotic process, the fusion pore, which may represent a different mechanism facilitating transmitter release.

Hypoxia induces voltage-dependent Ca2+ entry and quantal dopamine secretion in carotid body glomus cells.

We have investigated the changes of cytosolic [Ca2+] and the secretory activity in single glomus cells dispersed from rabbit carotid bodies during exposure to solutions with variable O2 tension (Po2). In normoxic conditions (Po2 = 145 mmHg; 1 mmHg = 133 Pa), intracellular [Ca2+] was 58 +/- 29 nM, and switching to low Po2 (between 10 and 60 mmHg) led to a reversible increase of [Ca2+] up to 800 nM. The response to hypoxia completely disappeared after removal of external Ca2+ or with the addition of 0.2 mM Cd2+ to the external solution. These same solutions also abolished both the Ca2+ current of the cells and the increase of internal [Ca2+] elicited by high external K+. Elevations of cytosolic [Ca2+] in response to hypoxia or to direct membrane depolarization elicited the release of dopamine, which was detected by amperometric techniques. Dopamine secretion occurred in episodes of spike-like activity that appear to represent the release from single secretory vesicles. From the mean charge of well-resolved secretory events, we estimated the average number of dopamine molecules per vesicle to be approximately 140,000, a value about 15 times smaller than a previous estimate in chromaffin granules of adrenomedullary cells. These results directly demonstrate in a single-cell preparation the secretory response of glomus cells to hypoxia. The data indicate that the enhancement of cellular excitability upon exposure to low Po2 results in Ca2+ entry through voltage-gated channels, which leads to an increase in intracellular [Ca2+] and exocytotic transmitter release.

Release of secretory products during transient vesicle fusion.

Patch-Camp experiments have shown that fusion of secretory granules with the plasma membrane does not always occur as an all-or-none event, but can develop slowly in a fluctuating manner or can be transient. These observations suggested that release could be detected during such incomplete fusion events. To test this hypothesis we have combined patch-clamp measurements of the activity of single exocytotic fusion pores in beige mouse mast cells with the electrochemical detection of serotonin released during the exocytotic events. We report here that on fusion pore opening there is a small release of serotonin which is directly proportional to the pore conductance. We also show that a significant release occurs during transient fusion events. These results demonstrate, to our knowledge for the first time, release of a neurotransmitter from a secretory vesicle that did not undergo complete fusion.

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.

Tension in secretory granule membranes causes extensive membrane transfer through the exocytotic fusion pore.

For fusion to occur the repulsive forces between two interacting phospholipid bilayers must be reduced. In model systems, this can be achieved by increasing the surface tension of at least one of the membranes. However, there has so far been no evidence that the secretory granule membrane is under tension. We have been studying exocytosis by using the patch-clamp technique to measure the surface area of the plasma membrane of degranulating mast cells. When a secretory granule fuses with the plasma membrane there is a step increase in the cell surface area. Some fusion events are reversible, in which case we have found that the backstep is larger than the initial step, indicating that there is a net decrease in the area of the plasma membrane. The decrease has the following properties: (i) the magnitude is strongly dependent on the lifetime of the fusion event and can be extensive, representing as much as 40% of the initial granule surface area; (ii) the rate of decrease is independent of granule size; and (iii) the decrease is not dependent on swelling of the secretory granule matrix. We conclude that the granule membrane is under tension and that this tension causes a net transfer of membrane from the plasma membrane to the secretory granule, while they are connected by the fusion pore. The high membrane tension in the secretory granule may be the critical stress necessary for bringing about exocytotic fusion.

Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells.

Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.

Compound versus multigranular exocytosis in peritoneal mast cells.

We have used the whole-cell patch-pipette technique to measure the step increases in the cell membrane capacitance (equivalent to the membrane area) caused by the fusion of secretory granules in degranulating murine mast cells. We have observed that up to 30% of the total membrane expansion caused by degranulation results from large fusion events that cannot be explained by the fusion of single secretory granules. These large events are observed mainly in the initial phase of a degranulation. We have developed a simple mathematical model for a mast cell to test whether these large events are caused by a stimulus-induced, granule-to-granule fusion that occurs before their exocytosis (multigranular exocytosis). Our results suggest that the large fusion events are caused by the exocytosis of granule aggregates that existed before stimulation and that are located at the cell's periphery. We propose a novel mechanism by which granule aggregates can be formed at the periphery of the cell. This mechanism relies on the ability of a transiently fused granule ("flicker") to fuse with more internally located granules in a sequential manner. This pattern may result in the formation of larger peripheral granules that later on can fuse with the membrane. The formation of peripheral granule aggregates may potentiate a subsequent secretory response.

Ionic basis of the differential neuronal activity of guinea-pig septal nucleus studied in vitro.

1. The electrical properties and ionic conductances of septal neurones were studied by intracellular recording in an in vitro slice preparation. Within the total number of cells recorded (n = 150) we identified three electrophysiological cell types, each one of them located in a separate septal region. Dorsolateral septal neurones comprised 60% of the cells, intermediate septal neurons 10%, and medical septal neurones 30%. 2. Passive electrical constants of dorsolateral, intermediate and medial septal neurones were, respectively:resting potential (-60.2 +/- 4.8, -59.8 +/- 3.3 and -56 +/- 4.3 mV); input resistance (82.5 +/- 17, 63 +/- 16 and 83 +/- 18 M omega) and membrane time constant (18.5 +/- 7.3, 14.2 +/- 6.8 and 10.7 +/- 3.4 ms). 3. Direct activation of dorsolateral septal neurones by current injection below 0.2 nA triggered repetitive firing of fast action potentials. Larger current pulses elicited a characteristic response consisting of an initial fast action potential followed by a train of slow spikes. An after-hyperpolarization followed termination of the pulse and the characteristic response. 4. In dorsolateral septal neurons tetrodotoxin (TTX) abolished the fast action potentials. The slow spikes and the after-hyperpolarization disappeared in presence of Co2+ or after brief removal of external Ca2+. This suggests that the characteristic response is mediated by Ca2+ and the after-hyperpolarization by a Ca2+-dependent K+ conductance. 5. The firing pattern of intermediate septal neurones activated from the resting potential spontaneously measured in the cells was similar to that of dorsolateral septal neurones; but direct activation from a hyperpolarized membrane potential evoked in intermediate septal cells a bursting response due to the generation of a low-threshold spike. The low-threshold spike was TTX-resistant but abolished by Co2+ and reached a maximal amplitude after hyperpolarization to -75 mV lasting for 100-150 ms. These results suggest the existence in intermediate septal neurons of a low-threshold Ca2+ conductance inactivated at the resting potential and deinactivated by hyperpolarization. 6. Depolarization of medial septal neurons by current pulses of amplitude greater than 0.2-0.3 nA elicited a typical burst of two to six action potentials. The bursts lasted for 20-50 ms and were followed by a marked after-hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological properties of guinea pig septal neurons in vitro.

The electrophysiological properties of septal neurons have been examined in vitro in guinea pig brain slices. These cells display different firing modes when stimulated by transmembrane current pulses depending on the amplitude of the depolarization. With small pulses septal neurons fire repetitive Na spikes but on larger depolarizations they respond with a single full-Na action potential which is followed by a number of spikes of smaller amplitude. A further increase in the amplitude of the pulse evokes powerful Ca spikes possibly generated in the dendrites. These Ca spikes appear with larger amplitude in presumptive intradendritic recordings. In many cells stimulation of the fimbria evoked postsynaptic responses consisting of either a depolarization, a hyperpolarization or a depolarization-hyperpolarization sequence.