Molecular mechanism of secretory vesicle docking

Docking, the stable association of secretory vesicles with the plasma membrane, is considered to be the necessary first step before vesicles gain fusion-competence, but it is unclear how vesicles dock. In adrenal medullary chromaffin cells, access of secretory vesicles to docking sites is controlled by dense F-actin (filamentous actin) beneath the plasma membrane. Recently, we found that, in the absence of Munc18-1, the number of docked vesicles and the thickness of cortical F-actin are affected. In the present paper, I discuss the possible mechanism by which Munc18-1 modulates cortical F-actin and how it orchestrates the docking machinery via an interaction with syntaxin-1. Finally, a comparison of Munc18's role in embryonic mouse and adult bovine chromaffin cell model systems will be made to clarify observed differences in cortical F-actin as well as docking phenotypes.

Molecular study of the Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

To identify the Na+/Ca2+ exchanger expressed in bovine chromaffin cells, the ncx gene was cloned from a bovine chromaffin cell cDNA library. Five partial clones were obtained and their nucleotide sequences showed that there were at least three isoforms containing different intracellular loops. The 3´-untranslated region was the same in all the clones. To examine the Na+/Ca2+ exchange activity of the clones, full-length ncx1 genes were constructed by replacing the corresponding region of bovine cardiac ncx1 clone p17 with the different regions from two bovine chromaffin cell clones; these were designated p17c and p17h. p17h, but not p17c, showed Na+/Ca2+ exchange activity when expressed in Chinese hamster ovary cells and Xenopus oocytes. The expressed exchange activity of p17 was inhibited by 8-bromoadenosine 3´:5´-cyclic monophosphate (8-Br-cAMP) but was not affected by PMA. However, the activity of p17h was inhibited by PMA but enhanced by 8-Br-cAMP. The agents that changed the activity of protein kinase C and cAMP-dependent protein kinase modulated the endogenous Na+/Ca2+ exchange current of chromaffin cells in a manner similar to that of p17h. Our results suggest that the p17h clone is the major isoform of the exchanger in chromaffin cells and is similar to the major ncx1 isoform in kidney. The exchange activity could be regulated by phosphorylation, and the variable region in the intracellular loop is important for the different effects of phosphorylation on the different isoforms.