Intracellular Ca ²⁺ signaling and store-operated Ca ²⁺ entry are required in Drosophila neurons for flight

Abstract

Neuronal Ca 2؉ signals can affect excitability and neural circuit formation. Ca 2؉ signals are modified by Ca 2؉ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca 2؉ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca 2؉ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP3R) can partially compensate for their loss of flight. Ca 2؉ measurements show that Orai gain-of-function contributes to the quanta of Ca 2؉ -release through mutant InsP3Rs and elevates store-operated Ca 2؉ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca 2؉ in neurons is required for Drosophila flight.calcium homeostasis ͉ flight patterns ͉ inositol 1,4,5-trisphosphate receptor ͉ sarco-endoplasmic reticulum-associated Ca 2ϩ ATPase ͉ STIM S everal aspects of neuronal function are regulated by ionic calcium (Ca 2ϩ ). Specific attributes of a Ca 2ϩ ''signature'' such as amplitude, duration, and frequency of the signal can determine the morphology of a neural circuit by affecting the outcome of cell migration, the direction taken by a growth-cone, dendritic development, and synaptogenesis (1). Ca 2ϩ signals also determine the nature and strength of neural connections in a circuit by specifying neurotransmitters and receptors (2). Most of these Ca 2ϩ signals have been attributed to the entry of extracellular Ca 2ϩ through voltage-operated channels or ionotropic receptors. However, other components of the ''Ca 2ϩ tool-kit'' coupled to Ca 2ϩ release from intracellular Ca 2ϩ stores are also present in neurons. These molecules include the store-operated Ca 2ϩ (SOC) channel, encoded by the Orai gene, identified recently in siRNA screens for molecules that reduce or abolish Ca 2ϩ influx from the extracellular milieu after intracellular Ca 2ϩ store depletion (3-5). Several reports have confirmed its identity as the pore forming subunit of the Ca 2ϩ -release activated Ca 2ϩ (CRAC) channel (6-8). Activation of this CRAC channel is mediated by the endoplasmic reticulum (ER) resident protein STIM (stromal interaction molecule), also identified in an RNAi screen for molecules that regulate SOC influx (9, 10). STIM senses the drop in ER Ca 2ϩ levels, and interacts with Orai by a mechanism which is only just being understood (11). Orai and STIM function in conjunction with the sarco-endoplasmic reticular Ca 2ϩ -ATPase pump (SERCA) to maintain ER store Ca 2ϩ and basal Ca 2ϩ . The importance of intracellular Ca 2ϩ homeostasis and SOC entry (E) in neural circuit formation and in neuronal function and physiology remains to be elucidated.Here, we report how Orai and STIM me...

Inositol 1,4,5-Trisphosphate Receptor and dSTIM Function inDrosophilaInsulin-Producing Neurons Regulates Systemic Intracellular Calcium Homeostasis and Flight

“…The construct was generated from a full-length dSTIM cDNA clone (BDGP LD45776). The other strains used are as follows: Dilp2GAL4, provided by Dr. E. Rulifson (Rulifson et al, 2002), embryonic wild-type itpr cDNA (UASitpr ϩ ) (Venkatesh et al, 2001), GAL80 ts with two inserts on the second chromosome (generated by Albert Chiang, NCBS, Bangalore, India), UASdOrai ϩ (Venkiteswaran and Hasan, 2009), DdcGAL4 referred in the text as aminergic (Li et al, 2000), a recombinant of Dilp2GAL4 and DdcGAL4 referred to as doubleGAL4 and UASmAChR (Venkiteswaran and Hasan, 2009). Elav C155 GAL4 (referred in the text as pan-neuronal), UASdicer(III) and UASmCD8GFP(II) were obtained from the Bloomington Stock Centre, Bloomington, IN.…”

“…Procedures for culturing Drosophila larval neurons have been described (Venkiteswaran and Hasan, 2009). Primary cultures of dissociated Drosophila larval neurons were plated in 200 l of Drosophila M1 medium (30 mM HEPES/150 mM NaCl/5 mM KCl/1 mM MgCl 2 /1 mM CaCl 2 /35 mM sucrose, pH 7.2) supplemented with 10% Fetal Bovine Serum (Invitrogen), 50 U/ml penicillin (Invitrogen), 50 g/ml streptomycin (Invitrogen), and 10 g/ml amphotericin B (Invitrogen) as described previously (Wu et al, 1983).…”

“…؉ in DILP2 or aminergic neurons suppresses flight defects of itpr mutants Certain adult flight phenotypes in itpr ka1091/ug3 , such as wing posture, spontaneous firing frequency and flight initiation can be partially suppressed by expression of the wild-type cDNA for the SOC dOrai ϩ in DILP2 and aminergic neurons (Venkiteswaran and Hasan, 2009). Together with the rescue by itpr ϩ expression in these domains and its requirement during pupal development, the data suggest that Ca 2ϩ release through the InsP 3 R and storeoperated Ca 2ϩ entry (SOCE) in DILP2 and aminergic neurons influence Drosophila flight circuit development to a significant extent.…”

“…We have previously shown that flight and associated defects observed in itpr gene (which encodes the only Drosophila InsP 3 R) mutants can be rescued by restoring InsP 3 R function in aminergic neurons (Banerjee et al, 2004) and that InsP 3 R-mediated Ca 2ϩ signaling is required at multiple steps for generating the neural circuit responsible for air-puff stimulated Drosophila flight (Banerjee et al, 2006). We also recently identified a requirement for SOCE in Drosophila neurons for regulating flight behavior (Venkiteswaran and Hasan, 2009).…”

The role of Ca2+ signaling on the self-renewal and neural differentiation of embryonic stem cells (ESCs)

“…has been demonstrated to contribute to the regulation of neural differentiation of ESCs as well as play a role in a variety of other neuronal activities. These include regulating the firing of the flight motor neurons in Drosophila melanogaster [78]; modulating the activity of the neuronal network in chronic epilepsy [79]; and regulating neuronal excitability, especially in injured neurons [80]. In addition, knockdown of STIM1 not only inhibited the differentiation of mouse Sox1-GFP 46C ESCs into neural progenitors, neurons and astrocytes but also caused severe cell death and suppresses the proliferation of neural progenitors during the early stage of neural differentiation [22].…”