Effects of Calcium and Calmodulin on the Binding of Rat Parotid Secretion Granules to the Plasma Membrane

Abstract

Since numerous studies suggest that Ca++ and calmodulin may modulate the fusion of secretion granules to the plasma membrane which takes place in exocytosis, we have examined the role of calcium and calmodulin in the binding of isolated parotid secretion granules to plasma membrane vesicles. 125I-labeled inside-out plasma membrane vesicles were incubated with secretion granules, the mixture was layered over 20% sucrose, the gradient was centrifuged, and the amount of 125I in the granule pellet was determined. Addition of Ca++ (20 nM to 10 pM) produced a concentration-dependent increase in the binding of 125I-labeled plasma membrane vesicles to the secretion granules, reaching a maximum value at 10 μM free Ca++; half-maximal binding occurred at 400 nM. Neither right-side-out parotid plasma membrane vesicles nor inside-out pancreatic islet plasma membrane vesicles bound to granules in the presence of 1 μM Ca++. Calmodulin produced a concentration-dependent increase in binding above that of Ca++ alone, and this effect was inhibited by the calmodulin antagonists, trifluoperazine and calmidazolium. Incubation of secretion granules with octadecylrhodamine B (R18)-loaded inside-out plasma membrane vesicles and 2 pM Ca++ caused de-quenching of fluorescence, indicating that the lipids in the granule membrane and the plasma membrane had intermixed. Added calmodulin increased the fluorescence two-fold above that with Ca ++ alone. These results suggest that Ca++ and calmodulin may play a role in parotid gland exocytosis by modulating the interaction between the secretion granules and plasma membrane.