Haller M, Heinemann C, Chow RH, Heidelberger R, Neher E

Haller M, Heinemann C, Chow RH, Heidelberger R, Neher E. spikes were much smaller (by 85%) and broader (by 3.5-fold) than those in control cells, suggesting that CAPS plays a role in determining release of vesicle contents via the fusion pore. Anti-CAPS IgGs also slowed the rate of the initial exocytotic capacitance burst, representing the docked-and-primed vesicle pool, by 90% but had no effect on the kinetics of rapid endocytosis. These results suggest that CAPS is a key component regulating the fusion of DCVs to the plasma membrane, and possibly fusion pore dilation, in catecholamine secretion from AC cells. L-cysteine gene unc-31 (Ann et al., 1997). Loss-of-function mutations in this gene result in pleiotropic nervous system abnormalities, suggesting that CAPS plays a fundamental role in neurosecretion in these invertebrates (Avery et al., 1993). Antibodies to CAPS are able to inhibit secretion in permeabilized PC12 cells (Walent et al., 1992; Ann et al., 1997) and selectively target catecholamine secretion in perforated rat brain synaptosomes (Tandon et al., 1998). Anti-CAPS IgG blocks norepinephrine secretion from PC12 cells and semi-intact synaptosomes but fails to antagonize glutamate secretion in the latter preparation. These findings indicate that CAPS plays a dedicated role in DCV but not SSV exocytosis. Although studies on permeabilized preparations strongly SIX3 suggest that CAPS is required for DCV exocytosis, they lack the L-cysteine kinetic resolution that can be obtained with electrophysiological analysis of intact cells. To further our understanding of the role of CAPS in secretion, we turned to patch-clamped calf AC cells. Secretion from these cells have been well characterized and can be recorded with millisecond resolution using either capacitance or amperometric techniques (Neher and Marty, 1982; Wightman et al., 1991; Chow et al., 1992; Artalejo et al., 1994; Elhamdani et al., 1998). We previously showed by using capacitance measurements that catecholamine secretion in these cells is preferentially coupled to a particular type of L-type Ca channel termed the facilitation Ca channel (Artalejo et al., 1994). More recent experiments using the high-resolution amperometric technique revealed that catecholamine secretion consists of two kinetic components (Elhamdani et al., 1998). Surprisingly, secretion elicited by activation of facilitation Ca channels is remarkably rapid (delay of 3 msec after the depolarization; termed strongly coupled secretion), approaching the speeds characteristic of synaptic transmission (Elhamdani et al., 1998). We attributed strongly coupled secretion to colocalization of facilitation Ca channels with DCV release sites. Slower secretion (delay of 25 msec or weakly coupled) is also observed, probably attributable to Ca channels that are not colocalized with the release apparatus (Klingauf and Neher, 1997;Elhamdani et al., 1998). Capacitance recordings also reveal multiple phases of secretion with an initial exocytotic burst, manifest as a very high initial rate of secretion, L-cysteine being caused by an already docked-and-primed release-ready pool of DCVs preceding a slower phase that may reflect secretion of newly recruited vesicles (Parsons et al., 1995). This kinetic diversity, along with the opportunity to analyze other parameters revealed by electrochemical analysis, such as the shape of unitary amperometric spikes, permits us in the present study to examine the effects of intracellular antagonism of CAPS on several aspects of catecholamine secretion in detail. The results suggest that CAPS plays a role at the very final step in DCV exocytosis where fusion of the vesicle with the membrane surface takes place. MATERIALS AND METHODS Cell?culture Bovine calf (average age 10C12 weeks) chromaffin cells were prepared by digestion of adrenal glands obtained from local slaughterhouses. Cells were purified and cultured using previously described methods (Artalejo et al., 1991). Cells plated at a density of 3 105 cells on collagen-coated 35-mm-diameter dishes were used in all studies, within 1 week of plating. Electrophysiology Our patch-clamp techniques have been published previously (Artalejo et al., 1995); an Axopatch 200 B (Axon Instruments, Foster City, CA) was used as the patch-clamp amplifier throughout these experiments. Capacitance was measured by a computer program using a phase-tracking technique. A standard protocol of ten 50 msec depolarizations from a holding potential of ?90 mV to +10 mV, each.