EXOCYTOSIS IN SINGLE CHROMAFFIN CELLS - REGULATION BY A SECRETORY GRANULE-ASSOCIATED GO PROTEIN

1, Besides having a role in signal transduction, trimeric G proteins m ay also be involved in membrane trafficking events, In chromaffin cell s, G alpha o has been found associated with the membrane of secretory granules, Here we examined the role of Go in regulated exocytosis usin g pressure microinjection combined with amperometric measurement of ca techolamine secretion from individual chromaffin cells, 2, Microinject ion of GTP gamma S and mastoparan strongly inhibits the amperometric r esponse to either nicotine or high K+. 3, The presence of mastoparan i n the cell incubation medium had no effect on K+-evoked secretion, sug gesting that mastoparan blocks the exocytotic machinery through an int racellular target protein not located just beneath the plasma membrane , 4, Microinjection of anti-C alpha o antibodies potentiates by more t han 50% the K+-evoked secretion, whereas anti-G alpha i(1/2) antibodie s have no effect, 5, Thus an inhibitory Go protein, probably associate d with secretory granules, controls exocytosis in chromaffin cells. Th e intracellular proteins controlling organelle-associated G proteins a re currently unknown, The neuronal cytosolic protein GAP-43 stimulates G alpha o in purified chromaffin granule membranes and inhibits exocy tosis in permeabilized cells. We show here that microinjection of a sy nthetic peptide corresponding to the domain of GAP-43 that interacts w ith Go inhibits secretion. We suggest that GAP-43 or a related sytosol ic protein controls the exocytotic priming step in chromaffin cells by stimulating a granule-associated Go protein.