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

Abstract

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.

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

“…hGH is stored in LDCV (Schweitzer and Kelly, 1985) and is released upon stimulation, serving as a marker for secretion in cotransfected cells (Holz et al, 2000). Plasmids encoding EEtagged wild-type PI3K-C2␣ (PI3K-C2␣-wt) and PI3K-C2␣ containing a point mutation in the catalytic domain (PI3K-C2␣-R1251P) were transfected in HEK cells where both proteins were expressed at comparable levels (Figure 4).…”

“…Because hGH is stored in secretory vesicles (Schweitzer and Kelly, 1985), this method has been successfully used to elucidate the role played in neurosecretion by many proteins of interest (Holz et al, 2000). The enhancement of evoked secretion following overexpression of wild-type PI3K-C2␣ suggests that PI3K-C2␣ activity controls the progression of LDCV toward a fusion-competent state.…”

“…In addition, phosphatidylinositol 4-kinase (PI4K), an integral membrane protein of chromaffin granules and synaptic vesicles, is required for their exocytosis (Wiedemann et al, 1996). Finally, the maintenance of a plasma membrane pool of PtdIns(4,5)P 2 was suggested to be important for the last step of exocytosis (Holz et al, 2000;Cremona and De Camilli, 2001;Osborne et al, 2001).…”

“…In addition, phosphatidylinositol 4-kinase (PI4K), an integral membrane protein of chromaffin granules and synaptic vesicles, is required for their exocytosis (Wiedemann et al, 1996). Finally, the maintenance of a plasma membrane pool of PtdIns(4,5)P 2 was suggested to be important for the last step of exocytosis (Holz et al, 2000;Cremona and De Camilli, 2001;Osborne et al, 2001).Phosphatidylinositol 3-kinases (PI3K) have recently been shown to be involved in a variety of cellular functions, including cell migration (Maffucci et al, 2005), axonal guidance (Ming et al, 1999), long-term depression (Daw et al, 2002), and programmed cell death (Brunet et al, 2001). 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).…”

High Ca2+-phosphate transfection efficiency enables single neuron gene analysis

“…1,2 Among these different methods, Ca 2+ -phosphate precipitation remains one of the most popular and cost-effective techniques when transfecting neurons. [3][4][5][6] Neurons are very sensitive cells and easy to die if microenvironment changes. Although liposome fusion and viral infection yield higher transfection rate, they also typically lead to higher neuronal toxicity than the Ca 2+ -phosphate transfection method.…”