Exocytosis in single chromaffin cells: regulation by a secretory granule-associated Go protein.
1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, G alpha o has been found associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells. 2. Microinjection of GTP gamma S and mastoparan strongly inhibits the amperometric response to either nicotine or high K+. 3. The presence of mastoparan in the cell incubation medium had no effect on K(+)-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane. 4. Microinjection of anti-G alpha o antibodies potentiates by more than 50% the K(+)-evoked secretion, whereas anti-G alpha i1/2 antibodies have no effect. 5. Thus an inhibitory Go protein, probably associated with secretory granules, controls exocytosis in chromaffin cells. The intracellular proteins controlling organelle-associated G proteins are currently unknown. The neuronal cytosolic protein GAP-43 stimulates G alpha o in purified chromaffin granule membranes and inhibits exocytosis in permeabilized cells. We show here that microinjection of a synthetic peptide corresponding to the domain of GAP-43 that interacts with Go inhibits secretion. We suggest that GAP-43 or a related cytosolic protein controls the exocytotic priming step in chromaffin, cells by stimulating a granule-associated Go protein.
Vitale
N
N
Institut National de la Santé et de la Recherche Médicale, Strasbourg, France.
Gonon
F
F
Thiersé
D
D
Aunis
D
D
Bader
M F
MF
D000906:Antibodies / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000502:physiology; D004594:Electrophysiology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D019922:GAP-43 Protein; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000276:immunology / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000502:physiology; D008845:Microinjections; D009419:Nerve Tissue Proteins / Q000502:physiology; D009538:Nicotine / Q000494:pharmacology; D010446:Peptide Fragments; D010455:Peptides; D011188:Potassium / Q000494:pharmacology; D014862:Wasp Venoms / Q000494:pharmacology
D000906:Antibodies; D002395:Catecholamines; D019922:GAP-43 Protein; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010455:Peptides; D014862:Wasp Venoms; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009538:Nicotine; C037500:mastoparan; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D011188:Potassium