Ca(2+)-dependent activator protein for secretion is critical for the fusion of dense-core vesicles with the membrane in calf adrenal chromaffin cells.
Calcium-dependent activator protein for secretion (CAPS) is a neural/endocrine cell-specific protein that has been shown to function at the Ca(2+)-dependent triggering step of dense-core vesicle (DCV) exocytosis in permeabilized PC12 cells. To evaluate the function of CAPS under physiological conditions, we introduced affinity-purified anti-CAPS IgGs into calf adrenal chromaffin (AC) cells via a patch pipette and tested the kinetics of catecholamine secretion using both amperometric and membrane capacitance techniques. The antibodies reacted with a single major approximately 145 kDa protein in AC cells based on immunoblot analysis. AC cells stimulated with sequential trains of action potentials at 7 Hz resulted in successive secretory episodes of equivalent magnitude. When either of two different anti-CAPS IgGs or their Fab fragments were present, a rapid and progressive inhibition of catecholamine release ensued to a maximum of >80%. The effect was specific because preabsorption of IgGs with the respective antigens ablated the inhibitory effect, and the IgGs had no effect on Ca currents. CAPS immunoneutralization not only reduced the number of amperometric spikes but markedly altered the kinetic characteristics of the residual events. The remaining spikes were much smaller (by 85%) and broader (by approximately 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 approximately 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.
Elhamdani
A
A
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Martin
T F
TF
Kowalchyk
J A
JA
Artalejo
C R
CR
D000200:Action Potentials / Q000502:physiology*; D000313:Adrenal Medulla / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology*; D005071:Evoked Potentials / Q000502:physiology; D005089:Exocytosis; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007074:Immunoglobulin G / Q000494:pharmacology; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology*; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D051381:Rats
D002135:Calcium-Binding Proteins; D002395:Catecholamines; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D002118:Calcium