Spontaneous Ca2+ Influx in Drosophila Pupal Neurons Is Modulated by IP3-Receptor Function and Influences Maturation of the Flight Circuit

Abstract

Inositol 1,4,5-trisphosphate receptors (IP3R) are Ca2+ channels on the neuronal endoplasmic reticulum (ER) membrane. They are gated by IP3, produced upon external stimulation and activation of G protein-coupled receptors on the plasma membrane (PM). IP3-mediated Ca2+ release, and the resulting depletion of the ER store, triggers entry of extracellular Ca2+ by store-operated Ca2+ entry (SOCE). Mutations in IP3R attenuate SOCE. Compromised IP3R function and SOCE during pupal development of Drosophila leads to flight deficits and mimics suppression of neuronal activity during pupal or adult development. To understand the effect of compromised IP3R function on pupal neuronal calcium signaling, we examined the effects of mutations in the IP3R gene (itpr) on Ca2+ signals in cultured neurons derived from Drosophila pupae. We observed increased spontaneous Ca2+ influx across the PM of isolated pupal neurons with mutant IP3R and also a loss of SOCE. Both spontaneous Ca2+ influx and reduced SOCE were reversed by over-expression of dOrai and dSTIM, which encode the SOCE Ca2+ channel and the ER Ca2+-sensor that regulates it, respectively. Expression of voltage-gated Ca2+ channels (cac, Ca-α1D and Ca-αT) was significantly reduced in itpr mutant neurons. However, expression of trp mRNAs and transient receptor potential (TRP) protein were increased, suggesting that TRP channels might contribute to the increased spontaneous Ca2+ influx in neurons with mutant IP3R. Thus, IP3R/SOCE modulates spontaneous Ca2+ influx and expression of PM Ca2+ channels in Drosophila pupal neurons. Spontaneous Ca2+ influx compensates for the loss of SOCE in Drosophila itpr mutant neurons.

Introduction

Inositol 1, 4, 5-trisphosphate receptors (IP3R) are Ca2+ channels on the neuronal endoplasmic reticulum (ER) membrane. IP3Rs are gated by the second messenger IP3, which is produced upon external stimulation and activation of G protein-coupled receptors (GPCRs) on the plasma membrane (PM). IP3-mediated Ca2+ release, and the resulting depletion of the ER store, triggers entry of extracellular Ca2+ by store-operated Ca2+ entry (SOCE). Drosophila mutants for the IP3R are flightless (Banerjee et al., 2004). Pan-neuronal knockdown of itpr (the gene for IP3R) and of genes encoding other calcium signaling molecules, such as GPCRs and the SOCE molecules, dSTIM and dOrai also result in flight deficits (Venkiteswaran and Hasan, 2009; Agrawal et al., 2010, 2013). These and other studies have shown that maturation of the flight circuit during pupal development requires intracellular calcium signaling through the IP3R followed by SOCE. This signaling is initiated by GPCRs and affects the transcriptional profile of developing flight circuit neurons (Agrawal et al., 2010, 2013; Pathak et al., 2015). Flight deficits in Drosophila with reduced intracellular calcium signaling during pupal stages, also correlate with reduced levels of Tyrosine Hydroxylase in dopaminergic neurons (Pathak et al., 2015), suggesting that calcium signaling through the IP3R in pupal neurons modulate neurotransmitter levels in adult Drosophila. In vertebrate neurons, neurotransmitter specification is modulated by calcium signaling through voltage-gated Ca2+ channels (VGCCs) and can be decoded by the frequency and amplitude of spontaneous Ca2+ transients (Spitzer et al., 2005; Dulcis et al., 2013); lower for excitatory neurotransmitters and higher for inhibitory neurotransmitters. Spontaneous Ca2+ signals (transients and sustained) in Drosophila pupal neurons are also mediated through VGCCs (Jiang et al., 2005; Iniguez et al., 2013). The TRPC class of PM Ca2+ channels function as polymodal cellular sensors and mediate changes in membrane voltage and intracellular calcium signals. In the developing Xenopus spinal cord TRPC channels are responsible for Ca2+ spike activity (Belgacem and Borodinsky, 2011). However a role for TRPC in generating spontaneous Ca2+ signals in Drosophila neurons is unknown.

Spontaneous Ca2+ oscillations in Drosophila occur in intact as well as in isolated brains, indicating that these signals are independent of sensory inputs (Rosay et al., 2001). In mushroom body Kenyon cells, frequency of spontaneous Ca2+ transients in isolated pupal neurons is similar to Ca2+ transients in vivo (Jiang et al., 2005). Here we have investigated the nature of spontaneous Ca2+ signals in cultured Drosophila pupal neurons where IP3/SOCE mediated intracellular Ca2+ signaling is disrupted. We show that pupal neurons from itpr mutants exhibit aberrantly high spontaneous Ca2+ influx and reduced SOCE. We propose that in itpr mutant neurons, higher spontaneous Ca2+ influx functions as a compensatory mechanism for decreased SOCE and helps maintain intracellular Ca2+ homeostasis. A possible source of the compensatory spontaneous Ca2+ influx appears to be the TRP channel.

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