Showing papers by "Manfred Lindau published in 2010"

Role of the synaptobrevin C terminus in fusion pore formation

Abstract: Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.

Push-and-pull regulation of the fusion pore by synaptotagmin-7

Abstract: In chromaffin cells, Ca2+ binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca2+ and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7–mutant chromaffin cells to analyze the role of Ca2+ binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca2+ binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca2+ binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.

Noradrenaline inhibits exocytosis via the G protein βγ subunit and refilling of the readily releasable granule pool via the αi1/2 subunit

Abstract: The molecular mechanisms responsible for the ‘distal’ effect by which noradrenaline (NA) blocks exocytosis in the β-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in INS 832/13 β-cells. NA inhibited Ca2+-evoked exocytosis by reducing the number of exocytotic events, without modifying vesicle size. Fusion pore properties also were unaffected. NA-induced inhibition of exocytosis was abolished by a high level of Ca2+ influx, by intracellular application of antibodies against the G protein subunit Gβ and was mimicked by the myristoylated βγ-binding/activating peptide mSIRK. NA-induced inhibition was also abolished by treatment with BoNT/A, which cleaves the C-terminal nine amino acids of SNAP-25, and also by a SNAP-25 C-terminal-blocking peptide containing the BoNT/A cleavage site. These data indicate that inhibition of exocytosis by NA is downstream of increased [Ca2+]i and is mediated by an interaction between Gβγ and the C-terminus of SNAP-25, as is the case for inhibition of neurotransmitter release. Remarkably, in the course of this work, a novel effect of NA was discovered. NA induced a marked retardation of the rate of refilling of the readily releasable pool (RRP) of secretory granules. This retardation was specifically abolished by a Gαi1/2 blocking peptide demonstrating that the effect is mediated via activation of Gαi1 and/or Gαi2.

Post-CMOS Fabrication of Working Electrodes for On-Chip Recordings of Transmitter Release

Abstract: The release of neurotransmitters and hormones from secretory vesicles plays a fundamental role in the function of the nervous system including neuronal communication. High-throughput testing of drugs modulating transmitter release is becoming an increasingly important area in the fields of cell biology, neurobiology, and neurology. Carbon-fiber amperometry provides high-resolution measurements of amount and time course of the transmitter release from single vesicles, and their modulation by drugs and molecular manipulations. However, these methods do not enable the rapid collection of data from a large number of cells. To allow this testing, we have developed a complementary metal-oxide semiconductor (CMOS) potentiostat circuit that can be scaled to a large array. In this paper, we present two post-CMOS fabrication methods to incorporate the electrochemical electrode material. We demonstrate by proof of principle the feasibility of on-chip electrochemical measurements of dopamine, and catecholamine release from adrenal chromaffin cells. The measurement noise is consistent with the typical electrode noise in recordings with external amplifiers. The electronic noise of the potentiostat in recordings with 400-?s integration time is ~ 0.11 pA and is negligible compared to the inherent electrode noise.

Hormonal inhibition of endocytosis: novel roles for noradrenaline and G protein Gz

Abstract: The modulation of endocytosis following exocytosis by noradrenaline (NA), a physiological inhibitor of insulin secretion, was investigated in INS 832/13 cells using patch-clamp capacitance measurements. Endocytosis was inhibited by NA in a pertussis toxin-insensitive manner. Dialysing a synthetic peptide mimicking the C-terminus of the α-subunit of Gz into the cells blocked the inhibition of endocytosis by NA. Cell-attached capacitance measurements indicated that inhibition by NA was due to a decreased number of endocytotic events without a change in vesicle size. Analysis of fission pore closure kinetics revealed two distinct fission modes, with NA selectively inhibiting the rapid fission pore closure events. Comparison of the actions of NA and deltamethrin, a calcineurin antagonist and potent inhibitor of endocytosis, demonstrated that they inhibit endocytosis by different mechanisms. These findings establish novel actions for NA and Gz in insulin-secreting cells and possibly other cell types.