Neurotransmitter release is modulated by many drugs and molecular manipulations. We present an active CMOS-based electrochemical biosensor array with high throughput capability (100 electrodes) for on-chip amperometric measurement of neurotransmitter release. The high-throughput of the biosensor array will accelerate the data collection needed to determine statistical significance of changes produced under varying conditions, from several weeks to a few hours. The biosensor is designed and fabricated using a combination of CMOS integrated circuit (IC) technology and a photolithography process to incorporate platinum working electrodes on-chip. We demonstrate the operation of an electrode array with integrated high-gain potentiostats and output time-division multiplexing with minimum dead time for readout. The on-chip working electrodes are patterned by conformal deposition of Pt and lift-off photolithography. The conformal deposition method protects the underlying electronic circuits from contact with the electrolyte that covers the electrode array during measurement. The biosensor was validated by simultaneous measurement of amperometric currents from 100 electrodes in response to dopamine injection, which revealed the time course of dopamine diffusion along the surface of the biosensor array. The biosensor simultaneously recorded neurotransmitter release successfully from multiple individual living chromaffin cells. The biosensor was capable of resolving small and fast amperometric spikes reporting release from individual vesicle secretions. We anticipate that this device will accelerate the characterization of the modulation of neurotransmitter secretion from neuronal and endocrine cells by pharmacological and molecular manipulations of the 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.TIR-FRET imaging | electrochemical imaging | time superresolution microscopy | image analysis | transmitter release N eurotransmitters, hormones, and many other mediators are stored in secretory vesicles, and their release occurs by the mechanism of exocytosis that begins with formation of a narrow fusion pore (1). Fusion-pore formation in neurosecretory vesicles is stimulated by an increase of intracellular [Ca 2+ ] and is thought to be induced by a large conformational change in the SNARE complex (2-4). Such changes may be involved in various steps from preparing vesicles for fusion (5) to fusion-pore dilation (6). To determine whether a conformational change in SNAREs is linked to fusion, the synaptosomal-associated protein 25 (SNAP25) mutant SCORE (SNARE COmplex REporter) has been developed (7), which contains two fluorescent proteins, CFP as a fluorescence resonance energy transfer (FRET) donor and Venus as a FRET acceptor. SCORE and constructs like it (5, 7-9) have the advantage that donor and acceptor exist at fixed stoichiometry, facilitating the analysis and interpretation of the measurements. Like SNAP25, SCORE forms SNARE complexes with syntaxin and vesicle-associated membrane protein (VAMP)/synaptobrevin (7,8), and when endogenous SNAP25 in chromaffin cells is cleaved by botulinum toxin E, a toxin-resistant SCORE rescues exocytosis (8). In PC12 cells expressing SCORE, a FRET change was evoked by high [K + ] stimulation, but with this method the time scale was tens of seconds (7). This FRET change was abolished in the absence of extracellular Ca 2+ , indicating that it is dependent on Ca 2+ entry. In contrast, the FRET change was not affected by tetanus neurotoxin treatment (7), which causes the blockade of exocytosis (10). In beta cells, a SCORE-like construct indicated a FRET chan...
SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules.Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25⌬9) showed changed fusion pore properties (Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events.
protein Von Willebrand factor (VWF), however, under specific conditions WPBs also contain a cocktail of small pro-inflammatory cytokines. WPB exocytosis is driven by an increase in intracellular free calcium ion concentration ([Ca 2þ ] i ); the majority of WPB fusion events result in complete discharge of cargo components, however, in a small fraction of cases cytokines are selectively released indicating that the fusion pore may act as a molecular size filter. Carbon fibre amperometry has been used to characterize fusion pore behavior in a number of cell types, but to date this approach has not been applied to WPBs. Therefore, we used this technique in combination with simultaneous optical imaging of fluorescent WPB exocytosis and changes in [Ca 2þ ] i and can report, for the first time, the kinetic properties of WPB fusion pore formation and expansion in human cultured endothelial cells. A clear delay (mean ~50 ms) is seen between the onset of the current spike and the increase in intra-WPB EGFP fluorescence indicating that WPB alkalinsation is delayed by the strong proton buffering capacity of the WPB lumen (55 mM/pH unit). Analysis of current spike parameters reveal a mean 25-75% rise time, peak amplitude and decay time of 1.63 ms, 50 pA and 6.63 ms respectively. Approximately 50% of current spikes were preceded by a foot signal of mean duration 6.34 ms. Occasional low amplitude, prolonged current increases, reminiscent of stand alone foot signals, were observed in conjunction with morphological rounding of WPBs, possibly reflecting kiss-and-run or lingering kiss fusion events. Following characterization of the WPB fusion pore under control conditions the impact of changing cellular parameters, including cholesterol levels, was also assessed.
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