Fusion pore expansion and contraction during catecholamine release from endocrine cells

Abstract

ABSTRACTAmperometry recording reveals the exocytosis of catecholamine from individual vesicles as a sequential process, typically beginning slowly with a pre-spike foot, accelerating sharply to initiate a spike, reaching a peak, and then decaying. This complex sequence reflects the interplay between diffusion, flux through a fusion pore, and possibly dissociation from a vesicle’s densecore. In an effort to evaluate the impacts of these factors, a model was developed that combines diffusion with flux through a static pore. This model recapitulated the rapid phases of a spike, but generated relations between spike shape parameters that differed from experimental results. To explore the possibility of fusion pore dynamics, a transformation of amperometry current was introduced that yields fusion pore permeability divided by vesicle volume (g/V). Applying this transform to individual fusion events yielded a highly characteristic time course.g/Vinitially tracks the pre-spike foot and the start of the spike, increasing ∼15-fold to the peak. However, after the spike peaks,g/Vunexpectedly declines and settles to a constant value that indicates the presence of a stable post-spike pore.g/Vof the post-spike pore varies greatly between events, and has an average that is ∼3.5-fold below the peak value and ∼4.5-fold above the pre-spike value. The post-spike pore persists andg/Vremains flat for tens of milliseconds, as long as catecholamine flux can be detected. Applying theg/Vtransform to rare events with two peaks revealed a stepwise increase ing/Vduring the second peak. Theg/Vtransform offers an interpretation of amperometric current in terms of fusion pore dynamics and provides a new framework for analyzing the actions of proteins that alter spike shape. The stable post-spike pore conforms with predictions from lipid bilayer elasticity, and offers an explanation for previous reports of prolonged hormone retention within fusing vesicles.STATEMENT OF SIGNIFICANCEAmperometry recordings of catecholamine release from single vesicles reveal a complex waveform with distinct phases. The role of the fusion pore in this waveform is poorly understood. A model based on a static fusion pore fails to recapitulate important aspects of the waveform. A new transform of amperometric current introduced here renders fusion pore permeability in real time. This transform reveals rich dynamic behavior of the fusion pore as catecholamine leaves a vesicle. This analysis shows that fusion pore permeability rapidly increases and then decreases before settling into a stable post-spike configuration.