Physiological significance of feedback regulation of inositol 1,4,5-trisphosphate receptor by cytoplasmic calcium

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The inositol 1,4,5-trisphosphate (IP_3) is a second messenger that is produced by hydrolysis of phosphatidyl-inositol 4,5-bisphosphate in response to activation by extracellular stimuli of the G protein- or tyrosine-kinase-coupled receptors on the plasma membrane. Using intrinsically IP_3R-deficient DT40 cells as the host cells for the receptor expression, we have tried to set up an experimental system in which functional analysis of recombinant IP_3 receptors (IP_3Rs) is practicable. After screening over 140 cell lines, we have established eleven stable cell lines that express mouse type 1 IP_3R (IP_3R1) with various contents. Both fluorescence immunocytochemical analysis and immunoelectron microscopic analysis showed that exogenously expressed IP_3R1 is localized on endoplasmic reticulum as native IP_3Rs. Stimulation of B cell receptor induced cytoplasmic Ca^<2+> increases in the mouse IP_3R1-expressing cells, indicating that the recombinant IP_3R1 in the stable cell lines functions properly. After reconstitution of membrane fractions prepared from these cell lines into planar lipid bilayers, we detected divalent cation currents. Single channel conductance, IP_3-dependency, and cytoplasmic Ca^<2+> dependency of the observed currents were similar to those of native IP_3R channels. Successful functional expression of IP_3R in the present expression system provides us an opportunity for detailed structure-functional characterization of IP_3R channels.