Biochemical characterization, distribution and phylogenetic analysis ofDrosophila melanogasterryanodine and IP3 receptors, and thapsigargin-sensitive Ca2+ ATPase

Abstract

IntroductionIntracellular Ca 2+ dynamics is a key factor in cellular signaling and physiology (for a review, see Bootman et al., 2001). In particular, the system of endomembranes that forms the sarco(endo)plasmic reticulum plays a vital role in Ca 2+ handling in most eukaryots (Carafoli and Klee, 1999). In this compartment two families of intracellular Ca 2+ release channels have been characterized: the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate receptors (IP3R) (Nori et al., 1993). Besides these, there is evidence for two more intracellular Ca 2+ releasing channels: the NAAPD and the sphingolipid receptors (Petersen and Cancela, 1999;Cancela, 2001). Ca 2+ re-uptake by the sarco(endo)plasmic reticulum is mediated by the thapsigargin-sensitive sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA), whose structure and function has been studied extensively in mammalian muscle systems (MacLennan, 1990).The development of the fruit fly Drosophila melanogaster is amenable to multidisciplinary analyses (for a review, see Campos-Ortega and Hartenstein, 1997) and is thus a powerful system in which to examine the role of these proteins in intracellular Ca 2+ homeostasis. In this organism, a single RyR gene with 26 exons, dry, and a single IP3R gene with 12 exons, dip, exist and have been genetically characterized (Takeshima et al., 1994;Sinha and Hasan, 1999). In contrast, RyR and IP3R in vertebrates are coded by at least three different genes that, due to alternative splicing, present a large number of isoforms (Rubtsov and Batrukova, 1997;Marks, 1997). A similar situation occurs with the thapsigargin-sensitive Ca 2+ ATPase. In D. melanogaster only one gene coding for this P-type ATPase has been detected, CaP60A (Magyar et al., 1995); whereas in vertebrates, at least three different isoforms of this Ca 2+ ATPase have been reported (Misquitta et al., 1999).Despite extensive genetic and molecular biology data for these proteins, there is a dearth of basic biochemical information on the Drosophila RyR, IP3R and SERCA proteins. In order to reap the benefit from a genetic and molecular biology tractable model organism with single RyR, IP 3 R and SERCA proteins, we characterize here these important molecules using Drosophila native endomembranes.

Synaptic neuropeptide release induced by octopamine without Ca 2+ entry into the nerve terminal

“…4B). To exclude off-target or developmental effects, ER Ca 2+ channels were acutely blocked by specific inhibitors known to be effective in Drosophila (23). Inhibiting RyRs with ryanodine (Ryan) or IP3Rs with xestospongin C (Xesto) blocked octopamine-induced neuropeptide release in the absence of extracellular Ca 2+ (Fig.…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…In C. elegans RyRs are found in body wall, vulval, anal, and pharyngeal muscles (Hamada et al 2002). In D. melanogaster a single isoform of RyR is expressed in digestive tract and nervous system (Vazquez-Martinez et al 2003).…”

Post-transcriptional Silencing and Functional Characterization of theDrosophila melanogasterHomolog of HumanSurf1

“…Caffeine contracture: contraction was induced by quickly immersing the preparation in physiological saline containing 25 mm caffeine. Caffeine causes maximal calcium release from the sarcoplasmic reticulum through the opening of ryanodine receptors (Vazquez-Martinez et al 2003). Whereas electrical stimulation (twitch and tetanus) induced only limited activations, caffeine induced a maximal contractile response; the amplitude of the caffeine contracture might be limited by the amount of calcium present in the intracellular stores or by the amount of myofibrillar proteins.…”