Dynasore inhibits rapid endocytosis in bovine chromaffin cells.

Vesicle recycling is vital for maintaining membrane homeostasis and neurotransmitter release. Multiple pathways for retrieving vesicles fused to the plasma membrane have been reported in neuroendocrine cells. Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To characterize its effects in modulating vesicle recycling and repetitive exocytosis, changes in the whole cell membrane capacitance of bovine chromaffin cells were recorded in the perforated-patch configuration. Constitutive endocytosis was blocked by dynasore treatment, as shown by an increase in membrane capacitance. The membrane capacitance was increased during strong depolarizations and declined within 30 s to a value lower than the prestimulus level. The amplitude, but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time 0 of the curve fitted to the decay phase were all approximately 110% of the membrane capacitance before stimulation, regardless of the dynasore concentration used. Membrane depolarization caused clathrin aggregation and F-actin continuity disruption at the cell boundary, whereas dynasore treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma membrane determined using atomic force microscopy was increased and the pore was wider in dynasore-treated cells. Our data indicate that dynamin-mediated endocytosis is the main pathway responsible for rapid compensatory endocytosis.

Ca2+ binding protein-1 inhibits Ca2+ currents and exocytosis in bovine chromaffin cells.

Calcium binding protein-1 (CaBP1) is a calmodulin like protein shown to modulate Ca2+ channel activities. Here, we explored the functions of long and short spliced CaBP1 variants (L- and S-CaBP1) in modulating stimulus-secretion coupling in primary cultured bovine chromaffin cells. L- and S-CaBP1 were cloned from rat brain and fused with yellow fluorescent protein at the C-terminal. When expressed in chromaffin cells, wild-type L- and S-CaBP1s could be found in the cytosol, plasma membrane and a perinuclear region; in contrast, the myristoylation-deficient mutants were not found in the membrane. More than 20 and 70% of Na+ and Ca2+ currents, respectively, were inhibited by wild-type isoforms but not myristoylation-deficient mutants. The [Ca2+]( i ) response evoked by high K+ buffer and the exocytosis elicited by membrane depolarizations were inhibited only by wild-type isoforms. Neuronal Ca2+ sensor-1 and CaBP5, both are calmodulin-like proteins, did not affect N(+, Ca2+ currents, and exocytosis. When expressed in cultured cortical neurons, the [Ca2+]( i ) responses elicited by high-K+ depolarization were inhibited by CaBP1 isoforms. In HEK293T cells cotransfected with N-type Ca2+ channel and L-CaBP1, the current was reduced and activation curve was shifted positively. These results demonstrate the importance of CaBP1s in modulating the stimulus-secretion coupling in excitable cells.