Regulated exocytosis triggered by the photolysis of a caged Ca2+ compound, DM-nitrophen, was investigated by patch-clamp capacitance measurements in two clones of PC12, the first wild-type and the second (PC12- 27) defective of both types of classical secretory vesicles together with the neuronal-type receptors for the attachment proteins of the N-ethylmaleimide- sensitive fusion protein, the so called SNAREs. Moreover, the electrophysiological data were correlated with the ultrastructure of resting quick-frozen-freeze-dried cells of the two clones. Wild-type PC12 exhibited two-component capacitance responses, time constants of 30-100 ms and >10 s, that previous studies had suggested to reflect primarily the fusion of the small and large secretory vesicles, each contributing cell surface increases of ≃10%. Both of these components were largely and specifically inhibited whether cells previously were microinjected with tetanus toxin light chain. In the defective clone, large responses also were recorded (≃19% surface expansion; time constant, ≃1 s) that, in contrast to those of the wild- type, were entirely resistant to the toxin. Although secretory organelles, i.e., large vesicles and also profiles of small vesicles, were abundant at the cell periphery and often docked to the plasmalemma of resting wild-type PC12, in the defective clone, no superficial accumulation of vesicles was observed. Our coordinate structural and functional results have revealed diversities between the two classical forms of regulated secretion in wild- type PC12 and have provided evidence of a toxin-insensitive form of Ca2+- induced exocytosis, prominent in the defective clone, that may play an important role(s) in cellular physiology.
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - Feb 2 1999|
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