Opening of individual exocytotic fusion pores in chromaffin cells was imaged electrochemically with high time resolution. Electrochemical detector arrays that consist of four platinum microelectrodes were microfabricated on a glass coverslip. Exocytosis of single vesicles containing catecholamines from a cell positioned on top of the array is detected by the individual electrodes as a time-resolved oxidation current, reflecting the time course of arrival of catecholamine molecules at the electrode surfaces. The signals exhibit low noise and reveal foot signals indicating fusion pore formation and expansion. The position of individual release events is determined from the fraction of catecholamines recorded by the individual electrodes. Simultaneous fluorescence imaging of release of acridine orange from individual vesicles confirmed the electrochemical position assignments. This electrochemical camera provides very high time resolution, spatiotemporal localization of individual fusion pore openings and quantitative data on the flux of transmitter from individual vesicles. Analysis of the amperometric currents employing random walk simulations indicates that the time course of amperometric spikes measured near the cell surface is due to a low apparent diffusion coefficient of cat-echolamines near the cell surface and not due to slow dissociation from the granular matrix.

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.

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.

High-throughput electrode arrays are required for advancing devices for testing the effect of drugs on cellular function. In this paper, we present design criteria for a potentiostat circuit that is capable of measuring transient amperometric oxidation currents at the surface of an electrode with submillisecond time resolution and picoampere current resolution. The potentiostat is a regulated cascode stage in which a high-gain amplifier maintains the electrode voltage through a negative feedback loop. The potentiostat uses a new shared amplifier structure in which all of the amplifiers in a given row of detectors share a common half circuit permitting us to use fewer transistors per detector. We also present measurements from a test chip that was fabricated in a 0.5-mum, 5-V CMOS process through MOSIS. Each detector occupied a layout area of 35 mumtimes15 mum and contained eight transistors and a 50-fF integrating capacitor. The rms current noise at 2-kHz bandwidth is ap110 fA. The maximum charge storage capacity at 2 kHz is 1.26times10⁶ electrons

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal transmembrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Application of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct application of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ∼70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex.