? AIterations in calcium (Ca) signaling are implicated in human pathologies ranging from neoplastic to neurodegenerative diseases. The spatio-temporal characteristics of Ca signals can regulate the activity of transcription factors and directly affect gene expression. Ca is generally mobilized from intracellular stores by inositol 1,4,5-trisphosphate (InsP3), which activates specific receptors containing an intrinsic Ca channel (InsP3Rs) and localized to the endoplasmic reticulum. Notably, many cell-surface receptors that increase InsP3 concurrently increase the intracellular production of sphingosine and sphingosine-1 phosphate (sphingosine-lP) as well. A Ca-mobilizing role for these two sphingolipids has been proposed, but their mechanism of action is unclear and the intracellular Ca channel(s) they activate yet to be identified. Furthermore, their functional interactions with InsP3 have not been characterized. The current proposal aims to test the hypothesis that both intracellular sphingosine and sphingosine-lP mobilize Ca through InsP3Rs.
The specific aims of this proposal are: 1) To characterize the Ca-mobilizing action of intracellular sphingosine and sphingosine-lP - acting separately or in combination with InsP3 - using caged-precursors that will be photo-activated inside living cells and their effects analyzed by single-cell microfluorimetry and digital imaging. The reciprocal interactions between InsP3 and the two sphingolipids during the Ca signaling following the stimulation of several plasma membrane receptors will also be investigated; 2) To establish whether the expression of different InsP3R-subtypes can determine the type of Ca signals evoked by intracellular sphingosine and sphingosine-lP. Experiments using cells genetically engineered to express specific InsP3R-subtypes will be combined with studies performed with mammalian cell lines transfected with distinct InsP3R-subtypes; 3) To ascertain whether sphingosine and sphingosine-lP directly open the InsP3R-channel and/or functionally modulate the action of InsP3. To this end, single-channel recording from native and recombinant InsP3R subtypes reconstituted in planar lipid bilayers will be employed. The long-term objective of this proposal is to identify the mechanisms and intracellular channels through which sphingosine and sphingosine-lP mobilize intracellular Ca - acting alone or in combination with InsP3 - and characterize their role in cellular signaling following the stimulation of plasma membrane receptors. ? ?
