Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca -evoked release of catecholamine

Moore, Johnnie M., Jason B. Papke, Anne L. Cahill, and Amy B. Harkins. Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca -evoked release of catecholamine. Am J Physiol Cell Physiol 291: C270–C281, 2006. First published February 8, 2006; doi:10.1152/ajpcell.00539.2005.—Synaptotagmin (syt) I is a Ca -binding protein that is well accepted as a major sensor for Ca -regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca and stimulated Ca influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca sensor for regulated release of transmitter.