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P#204

Dynamics of vesicular fusion in chromaffin cells – an amperometric view

Samuel Alberto Alfonso Bueno

  • Ciudad Autónoma de Buenos Aires,
  • Argentina
  • Samuel Alfonso-Bueno ¹
  • , Luciana I. Gallo ¹
  • , Fernando D. Marengo ¹
  • 1 Instituto de Fisiología, Biología Molecular y Neurociencias, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina

Chromaffin cells release adrenaline to the extracellular space through Ca2+-dependent exocytosis after fusion of secretory vesicles to the plasma membrane. Using amperometry, the dynamics of vesicular fusion can be recorded with sub-millisecond temporal resolution. A carbon fiber microelectrode is placed in close contact to the cell and, by applying an oxidation potential to the electrode, single-vesicle release of adrenaline can be detected as individual current spikes. Kinetic parameters, as amplitude (I), charge (quantal content, Q), half-width time (t1/2) and the decay time constant (τdecay), of the spike can be determined for each amperometric event. In this work, we evaluated the dynamics of vesicular fusion in mouse chromaffin cells by analyzing the amperometric spikes registered during stimulation with K+ 50 mM for 2 min. We analyzed the distribution of events according to Q and found that the Q1/3 distribution was best fit as the sum of three Gaussians after the comparison of one, two and three Gaussians fit with AIC and Fisher statistical tests. These vesicular subpopulations could also be described by the other kinetics parameters of the amperometric events when evaluated in relation to Q. These results strengthen the evidence for the presence of subpopulations of secretory vesicles. This analysis will be useful to determine key proteins regulating the dynamic of fusion of the different subpopulations of secretory vesicles in neurosecretory cells.

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