An Essential Role for a Small Synaptic Vesicle-Associated Phosphatidylinositol 4-Kinase in Neurotransmitter Release

Abstract

Glutamate release from nerve terminals is the consequence of Ca2+-triggered fusion of small synaptic vesicles with the presynaptic plasma membrane. ATP dependence of neurotransmitter release has been suggested to be founded, in part, on phosphorylation steps preceding membrane fusion. Here we present evidence for an essential role of phosphatidylinositol phosphorylation in stimulated release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes). Specifically, we show that a phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity resides on nerve terminal-derived small synaptic vesicles (SSVs) and that inhibition of the PtdIns 4-kinase activity in intact synaptosomes leads to attenuation of the evoked release of glutamate. The attenuation of transmitter release is reversible and correlates with respective changes in intrasynaptosomal PtdIns 4-kinase activity. Because only the Ca2+-dependent release of glutamate is affected, regulation appears to be at the level of exocytosis. Taken together, our data imply a mandatory role for PtdIns 4-kinase and phosphoinositide products in the regulated exocytosis of SSV in mammalian nerve terminals.

Phosphatidylinositol 4-kinase type IIα is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles

“…Our results confirm the existence of a PI 4-kinase activity on synaptic vesicles (19) and identify the enzyme responsible for this activity as PI4KII␣. We further show that this enzyme accounts for the bulk of the PI 4-kinase activity in a brain extract and is present at high concentration at synapses.…”

“…In vitro lipid phosphorylation assays on cytosol, immunoprecipitated kinases, or recombinant purified kinases were performed as described with minor modifications (18). In a typical assay, the reaction mixture (50 l) contained 30 mM Hepes (pH 7.4), 100 mM KCl, 2 mM MgCl 2 , 1 mM EDTA, [10][11][12][13][14][15][16][17][18][19][20] g of substrate lipid [synthetic short chain phosphoinositides (Echelon, Salt Lake City) or a brain-derived phosphoinositide mixture (Sigma P-6023)], and 0.2% Triton X-100. The reaction was initiated by addition of 40 M ATP (10 Ci of [␥-32 P]ATP per assay; 1 Ci ϭ 37 kBq) and performed for 10 min at 37°C.…”

Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

“…To rule out any long-term effect of PI3K activity on this process, chromaffin cells were treated for 24 h in culture medium with LY294002 (1 M) without significant consequence on evoked catecholamine release (see supplementary material S1). Together, these experiments seem to exclude any direct involvement of most subtypes of PI3K in neuroexocytosis, as previously established in neurosecretory cells and synaptosomes (Wiedemann et al, 1996;Martin et al, 1997;Wiedemann et al, 1998). However, one PI3K isoform, type II PI3K-C2␣, is much less sensitive to these EE-tagged PI3K-C2␣ was transiently expressed in COS cells and immunoprecipitated with anti-EE monoclonal antibodies followed by protein A-Sepharose.…”

“…However, a direct involvement of this class of enzymes and their lipid products in neuroexocytosis has been questioned Milosevic et al, 2005), based on little or no observed inhibition of synaptosomal or neurosecretory cell exocytosis by the PI3K inhibitors wortmannin and LY294002 (Wiedemann et al, 1996;Martin et al, 1997;Wiedemann et al, 1998). However, at the neuromuscular junction, wortmannin was shown to inhibit both spontaneous and evoked quantal neurotransmitter release (Hong and Chang, 1999).…”

Phosphoinositides in Constitutive Membrane Traffic

“…PtdIns4K activity is found on chromaffin granules and on small synaptic vesicles and is required for vesicle fusion (386,387). PtdIns4K activity is also found on vesicles containing the Glut4 glucose transporter isolated from muscle (190).…”