Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase.

Abstract

We characterized the biochemistry, distribution and phylogeny of Drosophila ryanodine (RyR) and inositol triphosphate (IP3R) receptors and the endoplasmic reticulum Ca2+-ATPase (SERCA) by using binding and enzymatic assays, confocal microscopy and amino acid sequence analysis. [3H]-ryanodine binding in total membranes was enhanced by AMP-PCP, caffeine and xanthine, whereas Mg2+, Ruthenium Red and dantrolene were inhibitors. [3H]-ryanodine binding showed a bell-shaped curve with increasing free [Ca2+], without complete inhibition at millimolar levels of [Ca2+]. [3H]-IP3 binding was inhibited by heparin, 2-APB and xestospongin C. Microsomal Ca2+-ATPase activity was inhibited by thapsigargin. Confocal microscopy demonstrated abundant expression of ryanodine and inositol triphosphate receptors and abundant Ca2+-ATPase in Drosophila embryos and adults. Ryanodine receptor was expressed mainly in the digestive tract and parts of the nervous system. Maximum parsimony and Neighbour Joining were used to generate a phylogenetic classification of Drosophila ryanodine and insitol triphosphate receptors and Ca2+-ATPase based on 48 invertebrate and vertebrate complete sequences. The consensus trees indicated that Drosophila proteins grouped with proteins from other invertebrates, separately from vertebrate counterparts. Despite evolutionary distances, our functional results demonstrate that Drosophila ryanodine and inositol triphosphate receptors and Ca2+-ATPase are reasonably similar to vertebrate counterparts. Our protein expression data are consistent with the known functions of these proteins in the Drosophila digestive tract and nervous system. Overall, results show Drosophila as a valuable tool for intracellular Ca2+ dynamics studies in eukaryotes.