Showing papers by "Erwin Neher published in 2002"

Emerging roles of presynaptic proteins in Ca++-triggered exocytosis.

Abstract: Molecules involved in late steps of neurotransmitter release at the synapse can be examined by noting the two speeds of the components of the exocytotic burst that are triggered by an increase in free Ca++. From studies of Ca++-dependent exocytosis of large dense-core vesicles in chromaffin cells, it seems that initiation of the SNARE complex is the molecular event underlying the priming process and that Munc13 acts as a priming factor by opening syntaxin. If synaptic mechanisms are similar, much could be learned from the molecular and kinetic studies that can be performed in chromaffin cells. The twinning of techiques from biophysics and molecular biology has led to remarkable progress in understanding the molecular mechanisms of synaptic transmission. Here we review the current picture of Ca++-triggered exocytosis, which has emerged from studies of a simple cellular model, the adrenal chromaffin cell. We discuss the molecular players that have been assigned a specific role in a particular step of this process and give a brief outlook on what these insights might tell us about mechanisms of short-term plasticity at classical synapses.

Protein kinase C-dependent phosphorylation of synaptosome-associated protein of 25 kDa at Ser187 potentiates vesicle recruitment.

Abstract: Activation of protein kinase C (PKC) constitutes a key event in the upregulation of secretory strength in neurons and neurosecretory cells during extensive stimulation, presumably by speeding up vesicle supply. However, the molecular targets and their mode of action remain elusive. We studied the only PKC-dependent phosphorylation site in the neuronal soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, Ser187, in synaptosome-associated protein of 25 kDa (SNAP-25). This phosphorylation site is located within the negatively charged C-terminal end of SNAP-25, which has been shown to be of critical importance in calcium-triggered exocytosis. We combined mutational studies that used overexpression in chromaffin cells with capacitance measurements and flash photolysis of caged calcium, allowing for high time resolution during both the stimulation and measurement of exocytosis. Overexpression of mutants simulating the phosphorylated form of Ser187 accelerated vesicle recruitment after the emptying of the releasable vesicle pools. Overexpression of mutants simulating the nonphosphorylated form, or block of PKC, impaired the refilling of the vesicle pools to similar extents. Biochemical studies verified the phosphorylation of a subpopulation of SNAP-25 after elevation of intracellular calcium concentrations. Some of the mutations led to a moderately decreased fast exocytotic burst component, which did not seem to be associated with the phosphorylation state of SNAP-25. Thus the C terminus of SNAP-25 plays a role for both fast exocytosis triggering and vesicle recruitment, and the latter process is regulated by PKC-dependent phosphorylation.

The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis

Abstract: Synchronous neurotransmission depends on the tight coupling between Ca2+ influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.

Separation of Presynaptic and Postsynaptic Contributions to Depression by Covariance Analysis of Successive EPSCs at the Calyx of Held Synapse

Abstract: Synaptic short-term plasticity is considered to result from multiple cellular mechanisms, which may include presynaptic and postsynaptic contributions. We have recently developed a nonstationary EPSC fluctuation analysis (Scheuss and Neher, 2001) to estimate synaptic parameters and their transient changes during short-term synaptic plasticity. Extending the classical variance–mean approach, a short train of stimuli is applied repetitively, and the resulting EPSCs are analyzed for means, variances, and covariances. This provides estimates of the quantal size and quantal content for each EPSC in the train, and furthermore, an estimate of the number of release sites. The latter is less sensitive to heterogeneity in the release probability than that of the variance–mean approach. Here, we applied this analysis to the calyx of Held synapse in brainstem slices of young rats (postnatal day 8–10). We found significant negative covariance in the amplitude of successive EPSCs in a train. The analysis showed that the 10-fold depression in the EPSC amplitude during 100 Hz trains at elevated extracellular Ca2+ concentration resulted from a 2.5-fold reduction in quantal size caused by postsynaptic AMPA receptor desensitization and saturation, and a fourfold reduction in quantal content, which was partially relieved by application of cyclothiazide. The number of release sites estimated by covariance analysis was ≈2000 and significantly larger than estimates from variance–mean parabolas.

Specificity emerges in the dissection of diacylglycerol- and protein kinase C-mediated signalling pathways

Abstract: Diacylglycerol- and Ca2+-mediated activation of protein kinase C (PKC) is regarded as a key triggering step in numerous cellular processes, from the regulation of gene transcription to the modulation of stimulus secretion coupling in hormone and neurotransmitter release. However, because of the ubiquitous and often redundant expression of the known PKC isoforms, the large number of identified PKC regulators and substrates, and the presence of alternative diacylglycerol and Ca2+ receptors that are also activated by commonly used PKC activators, the precise role of PKC activity in these processes and the identity of the relevant PKC isoforms and substrates have often remained elusive. Particularly in stimulus secretion coupling and the related process of synaptic transmission, the identification of steps at which PKC is involved has been difficult. This is because in many studies the physiological readout is a signal which is several steps downstream of the one modulated by PKC, and, at least until recently, reagents used to stimulate or block PKC have often been quite unspecific. In the analysis of synaptic transmission, for instance, many preparations allow only to record postsynaptic responses to presynaptic nerve stimulation. If a PKC effect is observed in such a system, a first question to be answered is whether postsynaptic sensitivity has changed or else presynaptic transmitter release. Even if in a suitable experimental paradigm an observed effect is shown to be caused by presynaptic PKC action, a key question remains: does PKC act on the electrical excitability of the nerve terminal or on the transmitter release process? Furthermore, in case of an answer in favor of the latter possibility, one would have to ask whether it is actually the release machinery of a given secretory vesicle that is altered or rather the regulation of the availability of release-ready vesicles, i.e., the …

Verfahren und vorrichtung zur spektral differenzierenden, bildgebenden messung von fluoreszenzlicht

Abstract: Die Erfindung bezieht sich auf ein Verfahren zur spektral differenzierenden, bildgebenden Messung von Fluoreszenzlicht, bei dem eine Fluorophore verschiedener Spezies enthaltende Probe mit Anregungslicht wenigstens eines durch seine spektralen Eigenschaften und/oder die Anregungszeit definierten Anregungskanals bestrahlt wird und das von der Probe emittierte Fluoreszenzlicht von wenigstens einem durch seine spektrale Detektionscharakteristik und/oder die Detektionszeit definierten Detektionskanal empfangen und in ein digitales Signal umgewandelt wird, wobei das digitale Signal zur weiteren Verarbeitung in einer Speichereinheit einer digitalen Datenverarbeitungsanlage gespeichert wird. Das erfindungsgemasse Verfahren zeichnet sich dadurch aus, dass die Eigenschaften mehrerer als spezifische Kombinationen jeweils eines Anregungs- und eines Detektionskanals definierter Messkanale vor Durchfuhrung der Messung gemass dem Ergebnis eines von einer Berechnungseinheit der digitalen Datenverarbeitungsanlage durchgefuhrten, mathematischen Optimierungsverfahrens, das die Fluoreszenzcharakteristiken wenigstens einiger der vom Benutzer in der Probe vermuteten Fluorophore berucksichtigt, automatisch eingestellt oder entsprechende Anweisungen zur manuellen Einstellung durch den Benutzer gegeben werden. Die Erfindung bezieht sich ausserdem auf eine Vorrichtung zur Durchfuhrung des erfindungsgemassen Verfahrens.