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doi: 10.1242/10.1242/jcs.00455

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

¹ Department of Molecular and Cellular Neurobiology, Neurobiology Institute, Campus UNAM-Juriquilla, Universidad Nacional Autónoma de México, Querétaro 76230, Mexico
² Department of Developmental Neurobiology and Neurophysiology, Neurobiology Institute, Campus UNAM-Juriquilla, Universidad Nacional Autónoma de México, Querétaro 76230, Mexico

^* Authors for correspondence (e-mail: juan{at}zool.unizh.ch; mdiaz{at}calli.inb.unam.mx)

Accepted 5 March 2003

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.

Key words: Calcium release channel, Intracellular calcium, Sequence analysis, Confocal microscopy, Drosophila

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