Functional Properties of the Drosophila melanogaster Inositol 1,4,5-Trisphosphate Receptor Mutants

Abstract

The inositol (1,4,5)-trisphosphate receptor (InsP 3R) is an intracellular calcium (Ca 2+) release channel that plays a crucial role in cell signaling. In Drosophila melanogaster a single InsP 3R gene ( itpr) encodes a protein (DmInsP 3R) that is ∼60% conserved with mammalian InsP 3Rs. A number of itpr mutant alleles have been identified in genetic screens and studied for their effect on development and physiology. However, the functional properties of wild-type or mutant DmInsP 3Rs have never been described. Here we use the planar lipid bilayer reconstitution technique to describe single-channel properties of embryonic and adult head DmInsP 3R splice variants. The three mutants chosen in this study reside in each of the three structural domains of the DmInsP 3R—the amino-terminal ligand binding domain (ug3), the middle-coupling domain (wc703), and the channel-forming region (ka901). We discovered that 1), the major functional properties of DmInsP 3R (conductance, gating, and sensitivity to InsP 3 and Ca 2+) are remarkably conserved with the mammalian InsP 3R1; 2), single-channel conductance of the adult head DmInsP 3R isoform is 89 pS and the embryonic DmInsP 3R isoform is 70 pS; 3), ug3 mutation affects sensitivity of the DmInsP 3Rs to activation by InsP 3, but not their InsP 3-binding properties; 4), wc703 channels have increased sensitivity to modulation by Ca 2+; and 5), homomeric ka901 channels are not functional. We correlated the results obtained in planar lipid bilayer experiments with measurements of InsP 3-induced Ca 2+ fluxes in microsomes isolated from wild-type and heterozygous itpr mutants. Our study validates the use of D. melanogaster as an appropriate model for InsP 3R structure-function studies and provides novel insights into the fundamental mechanisms of the InsP 3R function.