Influence of Cholesterol on Catecholamine Release from the Fusion Pore of Large Dense Core Chromaffin Granules

Abstract

Changes in cellular cholesterol can affect exocytosis, but the influence of cholesterol in fusion pore kinetics is unclear. Using carbon fiber amperometry, we monitored quantal catecholamine release from rat chromaffin cells. To bypass any possible effect of cholesterol perturbation on ion channels or the colocalization of voltage-gated Ca 2ϩ channels with sites of exocytosis, exocytosis was stimulated via uniform elevation of cytosolic [Ca 2ϩ ] (with whole-cell dialysis of a Ca 2ϩ -buffered solution). Under this condition, alterations of cellular cholesterol affected neither the mean number of amperometric events triggered per cell nor their quantal size and the kinetics of their main spike (which reflects the rapid release during and after rapid fusion pore dilation). In contrast, the reduction of cellular cholesterol shortened the "prespike foot" signals (which reflect the leakage of catecholamine via a semi-stable fusion pore) and reduced the proportion of "stand-alone foot" signals (which reflect the release via a flickering fusion pore that may close before it dilates significantly), whereas an oversupply of cholesterol had opposite effects. Acute extraction of cholesterol from the cytosol (via whole-cell dialysis of a cholesterol extractor) also shortened the prespike foot signals and reduced the proportion of stand-alone foot signals, but acute extracellular application of cholesterol extractor or "soluble" cholesterol had no effect. Our data raise the possibility that cholesterol molecules, particularly those in the cytoplasmic leaflet, helps to constrain the narrow waistline of a semi-stable fusion pore while it is flickering or before it starts to dilate rapidly.

Free Energy Landscape of Rim-Pore Expansion in Membrane Fusion

“…Further, the presence of molecules that oppose membrane rupture, such as, e.g., cholesterol, enhance survival of the nucleated hemifusion diaphragm and thereby facilitate expansion of the HD toward a more metastable size. Because the subsequent rupture of such an HD involves rim-pore formation, the presence of cholesterol is thus expected to enhance the propensity of pore-flickering (28). Finally, fluctuations in tension will discern between closure or complete opening of the rim-pore.…”

Exocytosis is impaired in mucopolysaccharidosis IIIA mouse chromaffin cells

“…The local lipid environment is strongly involved in regulating fusion pore formation, stability and the successful completion of exocytosis (Chasserot-Golaz et al, 2010). Membrane cholesterol has been shown to have an effect on chromaffin granule morphology (Tsuchiya et al, 2010), fusion pore kinetics and number of exocytotic events (Wang et al, 2010). Moreover, it has been shown that elevated cholesterol in b-cells causes reduced Ca 2+ entry (Lee et al, 2011).…”

The role of cholesterol in membrane fusion

“…Cholesterol’s direct effects on the dynamics of forming fusion pores has been measured in cells using amperometery (Ge et al, 2010; Wang et al, 2010; Koseoglu et al, 2011; Gruba et al, 2015; Finkenstaedt-Quinn et al, 2016). Recapituating these effects observed in cells, reconstitution of SNARE-mediated fusion in different biochemical settings also shows more efficient fusion when the cholesterol content is increased in the membrane (Tong et al, 2009; Kreutzberger et al, 2015; Stratton et al, 2016).…”