Cell Calcium 39 (2006) 187–196 Ectopic expression of a Drosophila InsP 3 R channel mutant has dominant-negative effects in vivo Sonal Srikanth, Santanu Banerjee, Gaiti Hasan ∗ National Centre for Biological Sciences, Tata Institute of Fundamental Research, Gandhi Krishi Vigyan Kendra Campus, Bangalore 560 065, India Received 9 December 2004; received in revised form 5 August 2005; accepted 26 October 2005 Available online 1 December 2005 Abstract The inositol 1,4,5-trisphosphate (InsP 3 ) receptor is a tetrameric intracellular calcium channel. It is an integral component of the InsP 3 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 InsP 3 R affects its functional properties, the kinetics of Ca 2+ -release in vitro from single point mutants of the Drosophila InsP 3 R 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 Ca 2+ upon stimulation as compared with cells over-expressing just the wild-type DmInsP 3 R, thus supporting our in vitro observations that increased Ca 2+ 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. © 2005 Elsevier Ltd. All rights reserved. Keywords: Ion selectivity filter; Muscarinic acetylcholine receptor; Aminergic neurons; Flight 1. Introduction Ionic calcium (Ca 2+ ) functions as an intracellular sig- naling molecule to regulate processes as diverse as fer- tilization, cell differentiation, acquisition and storage of memory, and cell death (reviewed in [3]). Mechanisms by which the concentration of free cytosolic Ca 2+ ([Ca 2+ ] i ) can undergo changes, include entry from the extracellular milieu and release from intracellular stores. The inositol 1,4,5-trisphosphate receptor (InsP 3 R) is a tetrameric ligand gated Ca 2+ channel present on membranes of intracellular calcium stores. It releases Ca 2+ into the cytosol in response to extracellular signals that generate InsP 3 . Unlike the mam- ∗ Corresponding author. Tel.: +91 80 23636421; fax: +91 80 23636462/662. E-mail address: gaiti@ncbs.res.in (G. Hasan). malian genome, which has three genes for the InsP 3 R, the Drosophila genome has a single gene for the InsP 3 R(itpr). This has allowed for the analysis of InsP 3 R function in vivo using well-characterized methods of Drosophila molecular genetics [4–6]. The Drosophila InsP 3 R is ∼60% homologous to the mammalian type I InsP 3 R, both of which comprise of an amino-terminal ligand binding domain, a central modula- tory domain and a carboxy-terminal channel domain. The structure–function relationships of the InsP 3 Rs have recently begun to be elucidated. Amino acid residues important for lig- and binding and modulation by ATP as well as regions respon- sible for Ca 2+ regulation and ion selectivity of the InsP 3 Rs have been identified (reviewed in [7]). Random single point mutants identified from genetic screens in Drosophila can provide important insights into the fundamental mechanisms of InsP 3 R function. In this context, the functional properties 0143-4160/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2005.10.013