Calcium-promoted fusion of isolated chromaffin granules detected by resonance energy transfer between labeled lipids embedded in the membrane bilayer

Abstract

Chromaffin granules isolated from bovine adrenal medulla were labeled with small unilamellar vesicles (SUV) made from N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and/or N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine as donor and acceptor fluorophores. Labeling of granules could be followed by the relief of self-quenching experienced when the labels were incorporated into the granule membrane. The mechanism of incorporation seemed to be fusion of the SUV with the granule membrane although transfer of label could not be ruled out. Granule-granule fusion could be detected either by resonance energy transfer from donor to acceptor fluorophores or by donor quenching in either of 2 different experimental designs: by a decrease in donor fluorescence and an increase in acceptor fluorescence when donor-labeled granules fused with acceptor-labeled granules or by an increase in donor fluorescence, and a decrease in acceptor fluorescence when granules containing both probes fused with unlabeled granules. Fusion of freshly prepared granules could be initiated by millimolar concentrations of Ca; Mg was less effective. Mg-ATP had no effect. Fusion was inhibited by potassium glutamate and a variety of organic and inorganic cations and anions, which also inhibited granule-granule aggregation to a lesser extent. The conditions for promotion and inhibition of granule-granule fusion were quite different from those reported by Knight and Baker for exocytosis of granule contents from permeabilized chromaffin cells. The membrane fusion seen here is probably activated by a different mechanism.