Spatiotemporal calcium signaling in a Drosophila melanogaster cell line stably expressing a Drosophila muscarinic acetylcholine receptor

Abstract

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca(2+)](i)) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca(2+)](i) transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.

Candidate Neural Substrates for Off-Edge Motion Detection in Drosophila

“…Vertebrate muscarinic receptors are coupled to G-proteins and various downstream signaling pathways to regulate a broad spectrum of cellular functions, including neuronal excitability [46]. In Drosophila , agonist activation of mAchR-A acts via the IP3 pathway to increase calcium release from internal stores [47], which elsewhere is reported in turn to activate high-conductance calcium-dependent potassium (BK) channels (the slowpoke channel), leading to membrane hyperpolarization [48]. It was therefore interesting that, using single-cell RT-PCR, we detected slowpoke transcripts in both T4 and T5 (Figure S1C), consistent with a previous immunohistochemical study that slowpoke is expressed in the optic lobe, including the lobula [49].…”

“…The coupling of the inhibitory inputs to secondary messenger system also provides a potential mechanism to adapt the temporal delay filter (τ a in Figure 5B; [50–52]). In addition, the activation of Drosophila mAchR-A in cultured cells also induces a secondary calcium influx, potentially originating from an extracellular calcium pool [47], while the activation of vertebrate muscarinic receptors has been shown to inhibit potassium channels, including those of the Kv7 (KCNQ/M) type, and to lower the excitability threshold (Figure 5B; [53, 54]). It is therefore possible that an interaction between the nicotinic and muscarinic inputs provides some form of multiplication, as suggested in the Hassenstein-Reichardt model (Figure 5B).…”

Molecular basis of the CRAC channel

“…The Drosophila S2 cell line attracted our attention because its utility for studying Ca 2+ signaling mechanisms was already known [19][20][21][22][23][24]. S2 cells were an attractive target for performing an RNAi-based screen to identify genes that controls SOCE because most fundamental cellular processes are conserved from Drosophila to mammals and the Drosophila genome had just been published earlier in 2000 [25].…”

A Genetic RNAi Screen for IP3/Ca2+ Coupled GPCRs in Drosophila Identifies the PdfR as a Regulator of Insect Flight

“…The mAcR can stimulate the IP 3 R in transfected S2 cells [17], [18], [33], [34] and by over-expression in primary neuronal cultures [4]. Similarly, the FmrfR was shown to modulate intracellular Ca 2+ in type 1 nerve terminals and thus regulate light-dependant escape behavior in Drosophila larvae [35].…”

“…Recent pharmacological evidence has implicated various monoamines such as octopamine, dopamine, tyramine and histamine (and presumably their receptors) and the muscarinic acetylcholine receptor (mAcR) in locust flight initiation [16]. The Drosophila mAcR increases IP 3 dependent intracellular Ca 2+ upon activation by its agonist in transfected S2 cells [17], [18] and in primary neuronal cultures from Drosophila [4]. Drosophila mutants that reduce octopamine levels exhibit flight initiation and maintenance defects which can be suppressed by pharmacological blocking of Tyramine receptors [19].…”