The Speech Of New York City.

PhDLanguageModern languageUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/179045/2/0001103.pd

Conformational Changes Induced in Voltage-gated Calcium Channel Cav1.2 by BayK 8644 or FPL64176 Modify the Kinetics of Secretion Independently of Ca2+ Influx*

The role of the L-type calcium channel (Cav1.2) as a molecular switch that triggers secretion prior to Ca2+ transport has previously been demonstrated in bovine chromaffin cells and rat pancreatic beta cells. Here, we examined the effect of specific Cav1.2 allosteric modulators, BayK 8644 (BayK) and FPL64176 (FPL), on the kinetics of catecholamine release, as monitored by amperometry in single bovine chromaffin cells. We show that 2 μm BayK or 0.5 μm FPL accelerates the rate of catecholamine secretion to a similar extent in the presence either of the permeable Ca2+ and Ba2+ or the impermeable charge carrier La3+. These results suggest that structural rearrangements generated through the binding of BayK or FPL, by altering the channel activity, could affect depolarization-evoked secretion prior to cation transport. FPL also accelerated the rate of secretion mediated by a Ca2+-impermeable channel made by replacing the wild type α11.2 subunit was replaced with the mutant α11.2/L775P. Furthermore, BayK and FPL modified the kinetic parameters of the fusion pore formation, which represent the initial contact between the vesicle lumen and the extracellular medium. A direct link between the channel activity and evoked secretion lends additional support to the view that the voltage-gated Ca2+ channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell

Distinct subcellular localization of a group of synaptobrevin-like SNAREs in Paramecium tetraurelia and effects of silencing SNARE-specific chaperone NSF

We have identified new synaptobrevin-like SNAREs and localized the corresponding gene products with green fluorescent protein (GFP)-fusion constructs and specific antibodies at the light and electron microscope (EM) levels. These SNAREs, named Paramecium tetraurelia synaptobrevins 8 to 12 (PtSyb8 to PtSyb12), showed mostly very restricted, specific localization, as they were found predominantly on structures involved in endoor phagocytosis. In summary, we found PtSyb8 and PtSyb9 associated with the nascent food vacuole, PtSyb10 near the cell surface, at the cytostome, and in close association with ciliary basal bodies, and PtSyb11 on early endosomes and on one side of the cytostome, while PtSyb12 was found in the cytosol. PtSyb4 and PtSyb5 (identified previously) were localized on small vesicles, PtSyb5 probably being engaged in trichocyst (dense core secretory vesicle) processing. PtSyb4 and PtSyb5 are related to each other and are the furthest deviating of all SNAREs identified so far. Because they show no similarity with any other R-SNAREs outside ciliates, they may represent a ciliate-specific adaptation. PtSyb10 forms small domains near ciliary bases, and silencing slows down cell rotation during depolarization-induced ciliary reversal. NSF silencing supports a function of cell surface SNAREs by revealing vesicles along the cell membrane at sites normally devoid of vesicles. The distinct distributions of these SNAREs emphasize the considerable differentiation of membrane trafficking, particularly along the endo-/phagocytic pathway, in this protozoan