Comparison of Secretory Responses as Measured by Membrane Capacitance and by Amperometry

• Published in: Biophysical journal Vol. 74; no. 4; pp. 2100 - 2113
• Main Authors: Haller, Mirjam, Heinemann, Christian, Chow, Robert H., Heidelberger, Ruth, Neher, Erwin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.1998
• Subjects: Adenosine Triphosphate - metabolism; Animals; Biophysical Phenomena; Biophysics; Calcium - metabolism; Cattle; Cell Degranulation - radiation effects; Chromaffin Cells - metabolism; Chromaffin Cells - radiation effects; Chromaffin Cells - secretion; Electric Conductivity; Electrochemistry; Exocytosis; In Vitro Techniques; Membrane Potentials; Models, Biological; Monte Carlo Method; Photolysis
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(98)77917-2

We have compared capacitance and amperometric measurements in bovine chromaffin cells when secretion was elicited by flash photolysis of caged-calcium or step depolarizations. Total amperometric charge depended linearly on the amount of capacitance increase in both types of experiments. Furthermore, the properties of resolvable amperometric spikes after flashes were comparable to those observed after depolarizations, and their timing was compatible with the rate of capacitance increase. For a more detailed comparison, we used Monte Carlo simulations of multiple amperometric events occurring randomly over the surface of a sphere and summing together, to generate a reference amperometric signal for a given measured capacitance increase. Even after correction for endocytotic processes, the time courses of the integrated experimental records lagged behind the integrated Monte Carlo records by ∼50 ms in flash and depolarization experiments. This delay was larger by ∼40 ms than what can be expected from the “pre-foot delay” or the foot duration. Possible sources for the remaining delay could be diffusional barriers like the patch-pipette and the chamber bottom, which are not taken into account in the model. We also applied a novel type of fluctuation analysis to estimate the relative quantum size of an amperometric event. On average the estimates from experimental amperometric traces, in both flash and depolarization experiments, were 3–5 times smaller than estimates from simulated ones. This discrepancy can be due to contributions to the amperometric current from small vesicles, preferred release from cellular regions orientated toward the chamber bottom, or abundance of “foot-only” events. In conclusion, amperometric signals in flash and depolarization experiments displayed similar delayed average time courses and a lower estimate for the relative quantum size compared to the modeled amperometric signals. However, individual amperometric spikes were in agreement with expectations derived from capacitance signals.