Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms.
Using the patch-clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12-myristate 13-acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated approximately 2-fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 microM also resulted in a 2-fold acceleration of pore expansion; this effect was not prevented by staurosporine, indicating that intracellular Ca2+ and activation of PKC accelerate fusion pore expansion via distinct mechanisms. However, fusion pores can expand fully even when PKC is inhibited. In contrast, the phosphatase inhibitor alpha-naphthylphosphate inhibits exocytotic fusion and slows fusion pore expansion. These results demonstrate that, subsequent to its formation, fusion pore expansion is under control of proteins subject to functional changes based on their phosphorylation states.
Scepek
S
S
Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Heidelberg, Germany.
Coorssen
J R
JR
Lindau
M
M
D000818:Animals; D002118:Calcium / Q000502:physiology*; D015550:Cell Degranulation / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D004804:Eosinophils / Q000187:drug effects / Q000502:physiology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006736:Horses; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D009281:Naphthalenes / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D009281:Naphthalenes; D009943:Organophosphorus Compounds; C014694:naphthyl phosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium