Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca2+-evoked release of catecholamine.
Synaptotagmin (syt) I is a Ca2+-binding protein that is well accepted as a major sensor for Ca2+-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 Ca2+ 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 Ca2+ and stimulated Ca2+ 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 Ca2+ sensor for regulated release of transmitter.
Dept. of Pharmacological and Physiological Science, St. Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.
Johnnie M
JM
Moore
Jason B
JB
Papke
Anne L
AL
Cahill
Amy B
AB
Harkins
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002395:Catecholamines / Q000378:metabolism*; D020868:Gene Silencing; D008561:Membrane Fusion / Q000502:physiology*; D016716:PC12 Cells; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism*
D002395:Catecholamines; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D002118:Calcium