Functional properties of the Drosophila melanogaster inositol 1, 4, 5-trisphosphate receptor mutants

Abstract

The inositol (1,4,5)-trisphosphate receptor (InsP₃R) is an intracellular calcium (Ca²⁺) release channel that plays a crucial role in cell signaling. In Drosophila melanogaster a single InsP₃R gene (itpr) encodes a protein (DmInsP₃R) that is ~60% conserved with mammalian InsP₃Rs. 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 DmInsP₃Rs have never been described. Here we use the planar lipid bilayer reconstitution technique to describe single-channel properties of embryonic and adult head DmInsP₃R splice variants. The three mutants chosen in this study reside in each of the three structural domains of the DmInsP₃R-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 DmInsP₃R (conductance, gating, and sensitivity to InsP₃ and Ca²⁺) are remarkably conserved with the mammalian InsP₃R1; 2), single-channel conductance of the adult head DmInsP₃R isoform is 89 pS and the embryonic DmInsP₃R isoform is 70 pS; 3), ug3 mutation affects sensitivity of the DmInsP₃Rs to activation by InsP₃, but not their InsP₃-binding properties; 4), wc703 channels have increased sensitivity to modulation by Ca²⁺; and 5), homomeric ka901 channels are not functional. We correlated the results obtained in planar lipid bilayer experiments with measurements of InsP₃-induced Ca²⁺ fluxes in microsomes isolated from wild-type and heterozygous itpr mutants. Our study validates the use of D. melanogaster as an appropriate model for InsP₃R structure-function studies and provides novel insights into the fundamental mechanisms of the InsP₃R function.