title: Measurement of exocytosis by amperometry in adrenal chromaffin cells: effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics.
abstract: We have used carbon-fibre amperometry to examine the kinetics of individual secretory granule fusion/release events in bovine adrenal chromaffin cells. Transfection with plasmids encoding the light chains of botulinum neurotoxins (BoNTs) was used to investigate the effects of cleavage of syntaxin or SNAP-25 on exocytosis. Expression of BoNT/C1 or BoNT/E inhibited the extent of exocytosis that was evoked by application of digitonin/Ca(2+) to permeabilise and stimulate single chromaffin cells. Following neurotoxin expression, the residual release events were no different from those of control cells in their magnitude and kinetics from analysis of the amperometric spikes. In contrast, activation of protein kinase C (PKC) resulted in a modification of the kinetics of single granule release events. Following phorbol ester treatment, the amperometric spikes showed a significant decrease in their total charge due to a decrease in their mean half-width with increases in the rate of the initial rise and also the fall to baseline of the spikes. These changes were prevented by pre-treatment with the PKC inhibitor bisindolylmaleimide. These results suggest that PKC regulates the rate of fusion pore expansion and also subsequent pore closure or granule retrieval. A PKC-mediated regulation of kiss-and-run fusion may, therefore, control the extent of catecholamine release from single secretory granules. The experimental approach used here may provide further information on the protein constituents and regulation of the fusion pore machinery.
authors.affiliation: The Physiological Laboratory, University of Liverpool, Crown Street, L69 3BX, Liverpool, UK.
authors.forename: M E
authors.initials: ME
authors.lastname: Graham
authors.forename: R J
authors.initials: RJ
authors.lastname: Fisher
authors.forename: R D
authors.initials: RD
authors.lastname: Burgoyne
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008561:Membrane Fusion / Q000187:drug effects* / Q000502:physiology; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D010957:Plasmids; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D001905:Botulinum Toxins; C096324:botulinum toxin type C; D013755:Tetradecanoylphorbol Acetate; C096326:botulinum toxin type E