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Am J Physiol Cell Physiol 297: C397-C406, 2009. First published June 10, 2009; doi:10.1152/ajpcell.00562.2008
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NERVOUS SYSTEM CELL BIOLOGY

Dynasore inhibits rapid endocytosis in bovine chromaffin cells

Chia-Chang Tsai,1 Chih-Lung Lin,2 Tzu-Lun Wang,2 Ai-Chuan Chou,2 Min-Yi Chou,2 Chia-Hsueh Lee,2 I-Wei Peng,2 Jia-Hong Liao,2 Yit-Tsong Chen,1,3 and Chien-Yuan Pan2,4

1Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan; 2Institute of Zoology, 3Department of Chemistry, and 4Department of Life Science, National Taiwan University, Taipei, Taiwan, Republic of China

Submitted 4 November 2008 ; accepted in final form 29 May 2009

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 ~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.

actin; atomic force microscope; calcium current; clathrin-mediated endocytosis; dynamin; FM4-64


Address for reprint requests and other correspondence: C.-Y. Pan, Institute of Zoology, National Taiwan Univ., Rm. 730, Life Science Bldg., 1 Roosevelt Rd., Sec. 4, Taipei 106, Taiwan (e-mail: cypan{at}ntu.edu.tw); Y.-T. Chen, Dept. of Chemistry, National Taiwan Univ., and Institute of Atomic and Molecular Sciences, Academia Sinica, P. O. Box 23-166, Taipei 106, Taiwan (e-mail: ytcchem{at}ntu.edu.tw)






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