Ectopic expression of a Drosophila InsP₃R channel mutant has dominant-negative effects in vivo

Abstract

The inositol 1,4,5-trisphosphate (InsP₃) receptor is a tetrameric intracellular calcium channel. It is an integral component of the InsP₃ signaling pathway in multicellular organisms, where it regulates cellular calcium dynamics in many different contexts. In order to understand how the primary structure of the InsP₃R affects its functional properties, the kinetics of Ca²⁺-release in vitro from single point mutants of the Drosophila InsP₃R have been determined earlier. Among these, the Ka901 mutant in the putative selectivity-filter of the pore is of particular interest. It is non-functional in the homomeric form whereas it forms functional channels (with altered channel properties) when co-expressed with wild-type channels [S. Srikanth, Z. Wang, H. Tu, S. Nair, M.K. Mathew, G. Hasan, I. Bezprozvanny, Functional properties of the Drosophila melanogaster inositol 1,4,5-trisphosphate receptor mutants, Biophys. J. 86 (2004) 3634-3646; S. Srikanth, Z. Wang, G. Hasan, I. Bezprozvanny, Functional properties of a pore mutant in the Drosophila melanogaster inositol 1,4,5-trisphosphate receptor. FEBS Lett. 575 (2004) 95-98]. Here we show that due to its changed functional properties the Ka901 mutant protein has dominant-negative effects in vivo. Cells expressing Ka901:WT channels exhibit much higher levels of cytosolic Ca²⁺ upon stimulation as compared with cells over-expressing just the wild-type DmInsP₃R, thus supporting our in vitro observations that increased Ca²⁺ release is a property of heteromeric Ka901:WT channels. Furthermore, ectopic expression of the Ka901 mutant channel in aminergic cells of Drosophila alters electrophysiological properties of a flight circuit and results in defective flight behavior.