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Concentrations of phosphatidylinositol 4,5-bisphosphate and inositol-1,4,5-trisphosphate within the distal segment of squid photoreceptors

, : Concentrations of phosphatidylinositol 4,5-bisphosphate and inositol-1,4,5-trisphosphate within the distal segment of squid photoreceptors. Visual Neuroscience 10(5): 921-929

Although inositol trisphosphate (InSP-3) is a key substance in phototransduction of invertebrate photoreceptors, its intracellular concentration remains unknown. The purpose of this study was to assay its concentration and the concentration of its precursor, phosphatidylinositol bisphosphate (PtdInsP-2), within squid photoreceptors. Rhabdomeric membranes were purified and their PtdInsP-2 content measured with a phosphate assay after the extracted phospholipids were deacylated and separated by ion-exchange chromatography. At least 75% of the total PtdInSP-2 found in the retinal homogenate was associated with the plasma membranes of the rhabdomeric microvilli, where PtdInSP-2 was 3.1 +- 0.7% of the total phospholipids, a level comparable to values published for rat brain. In terms of rhodopsin, microvillar membranes contained 3.7 +- 0.9 mol PtdInsP-2/mol rho. The InsP-3 content of living retinas was measured with a radioreceptor assay. The basal content of dark-adapted retinas was 0.15 +- 0.05 InsP-3/rho, equivalent to 30 +- 9 nmol/g tissue that is about twice that of rat brains. Flash illumination ( apprxeq 1ms in duration) that photoactivated 1% of rhodopsin increased the level about fivefold to 0.68 +- 0.22 InsP-3/rho. Corresponding decrease in PtdInsP-2 was undetectable as it was within measurement errors. For PtdInsP-2, the measured content corresponds to 5.6 +- 1.4 mM within the volume of rhabdomere. Maximal light-induced concentration of InsP-3 is calculated to be 1.2 +- 0.4 mM within the cytoplasm of the distal segment. Each photoactivated rhodopsin leads to the formation of ltoreq 500 InsP-3 molecules when measured 15 s after the flash. Thus, high concentration of InsP-3 in these cells is primarily due to restricted intracellular volumes rather than to high amplification by the enzyme cascade. The InsP-3 concentration within squid photoreceptors is the highest yet reported for any transducing cell and may indicate the involvement of relatively low affinity receptors or channels during invertebrate phototransduction.

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