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Published 23 July 2001. doi:10.1083/jcb.200102098
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© The Rockefeller University Press, 0021-9525/2001/7/355 $5.00
The Journal of Cell Biology, Volume 154, Number 2, July 23, 2001 355-368

Article

Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity



Kristina D. Micheva1, Ronald W. Holz2 and Stephen J. Smith1

1 Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305
2 Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109

Address correspondence to Kristina D. Micheva, Department of Molecular and Cellular Physiology, Stanford University, 279 Campus Dr., Stanford, CA 94305. Tel.: (650) 725-7552. Fax: (650) 725-8021. E-mail: kmicheva{at}stanford.edu

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-{delta}1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.

Key Words: phosphatidylinositol 4,5-biphosphate; green fluorescent protein; synapse; NMDA; nitric oxide


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