A pleckstrin homology domain specific for phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis.
Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells.
Holz
R W
RW
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA. holz@umich.edu
Hlubek
M D
MD
Sorensen
S D
SD
Fisher
S K
SK
Balla
T
T
Ozaki
S
S
Prestwich
G D
GD
Stuenkel
E L
EL
Bittner
M A
MA
D000804:Angiotensin II / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D019439:Chromaffin Cells / Q000502:physiology*; D002837:Chromaffin Granules / Q000502:physiology*; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology; D005089:Exocytosis; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D019382:Human Growth Hormone / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains
D008164:Luminescent Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D000804:Angiotensin II; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; D004246:Dimethylphenylpiperazinium Iodide