Qinghua Fang

The role of the C terminus of the SNARE protein SNAP-25 in fusion pore opening and a model for fusion pore mechanics

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. It has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25Δ9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25Δ9 displayed smaller amperometric “foot-current” currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25Δ9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.

F-actin and myosin II accelerate catecholamine release from chromaffin granules.

The roles of nonmuscle myosin II and cortical actin filaments in chromaffin granule exocytosis were studied by confocal fluorescence microscopy, amperometry, and cell-attached capacitance measurements. Fluorescence imaging indicated decreased mobility of granules near the plasma membrane following inhibition of myosin II function with blebbistatin. Slower fusion pore expansion rates and longer fusion pore lifetimes were observed after inhibition of actin polymerization using cytochalasin D. Amperometric recordings revealed increased amperometric spike half-widths without change in quantal size after either myosin II inhibition or actin disruption. These results suggest that actin and myosin II facilitate release from individual chromaffin granules by accelerating dissociation of catecholamines from the intragranular matrix possibly through generation of mechanical forces.

Role of the synaptobrevin C terminus in fusion pore formation.

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.

Improved surface-patterned platinum microelectrodes for the study of exocytotic events.

Surface-patterned platinum microelectrodes insulated with 300 nm thick fused silica were fabricated using contact photolithography. These electrodes exhibit low noise and were used for monitoring single vesicle exocytosis from chromaffin cells by constant potential amperometry as well as fast-scan cyclic voltammetry. Amperometric spike parameters were consistent with those obtained with conventional carbon fiber electrodes. Catecholamine voltammograms acquired with platinum electrodes exhibited redox peaks with full width at half-maximum of approximately 45 mV, much sharper than those of carbon fiber electrode recordings. The time course of voltammetrically measured release events was similar for platinum and carbon fiber electrodes. The fused-silica-insulated platinum electrodes could be cleaned and reused repetitively and allowed incorporation of micrometer precision surface-patterned poly-D-lysine. Poly-D-lysine-functionalized devices were applied to stimulate mast cells and record single release events without serotonin preloading. Microfabricated platinum electrodes are thus able to record single exocytotic events with high resolution and should be suitable for highly parallel electrode arrays allowing simultaneous measurements of single events from multiple cells.

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

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.

How Could SNARE Proteins Open a Fusion Pore

The SNARE (Soluble NSF Attachment protein REceptor) complex, which in mammalian neurosecretory cells is composed of the proteins synaptobrevin 2 (also called VAMP2), syntaxin, and SNAP-25, plays a key role in vesicle fusion. In this review, we discuss the hypothesis that, in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains.

Transparent electrode materials for simultaneous amperometric detection of exocytosis and fluorescence microscopy.

We have developed and tested transparent microelectrode arrays capable of simultaneous amperometric measurement of oxidizable molecules and fluorescence imaging through the electrodes. Surface patterned microelectrodes were fabricated from three different conducting materials: Indium-tin-oxide (ITO), nitrogen-doped diamond-like carbon (DLC) deposited on top of ITO, or very thin (12-17 nm) gold films on glass substrates. Chromaffin cells loaded with lysotracker green or acridine orange dye were placed atop the electrodes and vesicle fluorescence imaged with total internal reflection fluorescence (TIRF) microscopy while catecholamine release from single vesicles was measured as amperometric spikes with the surface patterned electrodes. Electrodes fabricated from all three materials were capable of detecting amperometric signals with high resolution. Unexpectedly, amperometric spikes recorded with ITO electrodes had only about half the amplitude and about half as much charge as those detected with DLC or gold electrodes, indicating that the ITO electrodes are not as sensitive as gold or DLC electrodes for measurement of quantal catecholamine release. The lower sensitivity of ITO electrodes was confirmed by chronoamperometry measurements comparing the currents in the presence of different analytes with the different electrode materials.

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.

Both Gi and Go Heterotrimeric G Proteins Are Required to Exert the Full Effect of Norepinephrine on the β-Cell KATP Channel

The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (KATP) in INS 832/13 cells. Membrane potential was monitored under the whole cell current clamp mode. NE hyperpolarized the cell membrane, an effect that was abolished by tolbutamide. The effect of NE on KATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the KATP channels ∼2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the KATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric Gi/Go proteins. To identify the G proteins involved, antisera raised against α and β subunits (anti-Gαcommon, anti-Gβ, anti-Gαi1/2/3, and anti-Gαo) were used. Anti-Gαcommon totally blocked the effects of NE on membrane potential and KATP channels. Individually, anti-Gαi1/2/3 and anti-Gαo only partially inhibited the action of NE on KATP channels. However, the combination of both completely eliminated the action. Antibodies against Gβ had no effects. To confirm these results and to further identify the G protein subunits involved, the blocking effects of peptides containing the sequence of 11 amino acids at the C termini of the α subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that Gαi3 and Gαo1 were not involved. In conclusion, both Gi and Go proteins are required for the full effect of norepinephrine to activate the KATP channel.

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

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.