The Interactive Fly
Zygotically transcribed genes
Phylogenetic analysis reveals AMPA, kainate, and NMDA receptor families in insect genomes, suggesting conserved functional properties corresponding to their vertebrate counterparts. However, heterologous expression of the Drosophila kainate receptor DKaiR1D and the AMPA receptor DGluR1A revealed novel ligand selectivity at odds with the classification used for vertebrate glutamate receptor ion channels (iGluRs). DKaiR1D forms a rapidly activating and desensitizing receptor that is inhibited by both NMDA and the NMDA receptor antagonist AP5; crystallization of the KaiR1D ligand-binding domain reveals that these ligands stabilize open cleft conformations, explaining their action as antagonists. Surprisingly, the AMPA receptor DGluR1A shows weak activation by its namesake agonist AMPA and also by quisqualate. Crystallization of the DGluR1A ligand-binding domain reveals amino acid exchanges that interfere with binding of these ligands. The unexpected ligand-binding profiles of insect iGluRs allows classical tools to be used in novel approaches for the study of synaptic regulation (Li, 2016). Video Abstract
Glutamate is the major excitatory neurotransmitter in the vertebrate CNS; its actions are mediated largely via three classes of ionotropic glutamate receptors (iGluRs) named AMPA, kainate, and NMDA receptors. The classification of iGluRs into AMPA, kainate, and NMDA receptors was based on the efforts of medicinal chemists who identified subtype selective heterocyclic amino acids such as AMPA, kainate, and quisqualate and amino acid analogs such as NMDA and 2(R)-amino-5-phosphonopentanoic acid (D-AP5) that act as agonists and antagonists. This work was so successful that the selective action of NMDA and D-AP5 formed the corner stone on which the role of NMDA receptors in synaptic plasticity was established (Li, 2016).
Subsequent cloning of insect iGluRs, which revealed sequence similarity with their vertebrate AMPA, kainate, and NMDA receptor counterparts, suggests that the same series of ligands can be used to investigate their role in CNS function. However, with the exception of the neuromuscular junction (NMJ) of larval Drosophila and the NMJ of adult locusts, the small size and inaccessibility of insect neurons has to date challenged characterization of the functional properties of native insect iGluRs. Sequence analysis of the Drosophila genome identified 14 iGluR genes that resemble vertebrate AMPA, kainate, and NMDA receptors. Transcript profiling revealed that nine of these iGluRs are expressed in the brain, with five expressed at the neuromuscular junction. Very little is known about the structure and functional properties of Drosophila iGluRs and only recently was a functional reconstitution achieved for recombinant Drosophila NMJ iGluRs (Han, 2015). As a result, iGluRs are understudied in model organisms like Drosophila for which powerful genetic techniques have otherwise yielded numerous insights into the molecular neurobiology of synapse development and function (Li, 2016).
Four presumptive Drosophila kainate receptors (Clumsy, DKaiR1C, DKaiR1D, and CG11155) are functionally required for spectral preference behavior and are thought to mediate excitatory synaptic transmission from the second-order neuron Dm8 to the third-order neuron Tm5c (Karuppudurai, 2014). The eye-enriched kainate receptor (EKAR) is expressed in photoreceptors, receiving feedback glutamatergic signals from amacrine cells, but so far, in vitro reconstitution has not been achieved for any of these presumptive kainate receptors. Instead, functional analysis of their role in CNS glutamatergic circuits relies solely on chronic inactivation using genetic mutants and RNAi-mediated knockdown. This study combined electrophysiological, biochemical, and crystallographic analyses to determine receptor activity and ligand specificity of a Drosophila kainate receptor DKaiR1D and a Drosophila AMPA receptor DGluR1A. DKaiR1D was found to form functional homomeric channels in HEK cells and oocytes with pharmacological properties distinct from vertebrate and Drosophila NMJ iGluRs. Crystal structures of DKaiR1D ligand-binding dimer complexes with glutamate, NMDA, and AP5 revealed that only glutamate triggers domain closure and that NMDA and AP5 are antagonists. DGluR1A receptors respond weakly to AMPA and quisqualate; the crystal structure of DGluR1A revealed that the binding of these ligands is hindered by steric occlusion. Thus, despite structural and sequence similarity between insect and vertebrate iGluRs, insect iGluRs do not conform to the pharmacology-based classification of vertebrate iGluRs. However, the agonist/antagonist binding properties of insect iGluRs we report here provide a new approach for acute inactivation/activation in vivo and for dissecting their functions in complex neural circuits (Li, 2016).
This study found that DGluR1A and DKaiR1D, similar to vertebrate GluA1-4 AMPA and GluK1-3 kainate receptor subunits, form homomeric calcium-permeable channels. Based on sequence alignments and the lack of RNA-editing of Drosophila iGluRs mRNA at their Q/R sites, it is likely that most insect iGluRs are calcium permeable and that they are inhibited by endogenous cytoplasmic polyamines and by spider venom polyamine toxins. It is noted that homomeric DKaiR1D has a very fast desensitization rate, while for DGluR1A, fit has not yet been possible to achieve sufficient expression to allow recording from outside-out patches with rapid perfusion. Structural analyses revealed that DKaiR1D LBD dimers contain conserved Na+ ion binding sites characteristic of vertebrate kainate receptors, but these appear to not strongly modulate the activation or desensitization of KaiR1D, perhaps because the Cl- binding site found in vertebrate kainate receptors is absent in insect kainate receptors. Sequence analysis revealed that this separation of Na+ and Cl- binding sites in KaiR1D subunits occurs in all insect species examined. Structure-aided sequence analysis also reveals that in the other three groups of fly kainate receptors, different combinations of amino acid substitutions destroy or significantly weaken both the Na+ and Cl− binding sites. Thus, the allosteric modulation by both anions and cations that is characteristic of vertebrate kainate receptors is uncoupled in insect kainate receptors and for the majority of cases both ion binding sites are eliminated (Li, 2016).
Previous phylogenetic studies suggest that most bilateria, including insects, worms, and vertebrates, have three major classes of cation-selective iGluRs, corresponding to vertebrate AMPA, kainate, and NMDA receptors. The current analysis reveals that in insects, the kainate receptor family is expanded into four groups, while a prior phylogenetic analysis revealed that in Mollusca the AMPA receptor family is expanded. At the neuromuscular junction of Drosophila and the locust Schistocerca gregaria, iGluRs have been extensively studied, in part serving as a surrogate model for CNS iGluRs. Interestingly, this study found that late in evolution, in higher Diptera, the five Drosophila NMJ iGluR subunits, GluRIIA-E, were derived from two separate kainate receptor subtypes, KaiR1C and Clumsy. Thus, despite their unique obligate heterotetrameric subunit stoichiometry and insensitivity to kainate), fly NMJ iGluRs evolved from ancestral kainate-sensitive iGluRs, and it is likely that in other insect species iGluRs related to KaiR1C and Clumsy may function in both the CNS and NMJ (Li, 2016).
Although phylogenetic analysis supports classification of Drosophila and other insect iGluRs into the familiar AMPA, kainate, and NMDA receptor families, the current results reveal unexpected differences in their ligand-binding properties. The most dramatic change was the conversion of NMDA from an agonist for vertebrate NMDA receptors to an antagonist for Drosophila KaiR1D, a kainate receptor that is also inhibited by both isomers of AP5, while D-AP5, but not L-AP5 acts as a potent vertebrate NMDA receptor antagonist. The crystal structures solved in this study for the DKaiR1D LBD establish that NMDA and AP5 inhibit activation of DKaiR1D by stabilizing an open cleft conformation, similar to the action of competitive antagonists for vertebrate iGluRs from each of the three major families. In addition, NMDA triggered separation of the upper lobes of the DKaiR1D LBD dimer assembly, a conformational change that occurs during desensitization of vertebrate AMPA and kainate receptors, may in addition contribute to the inhibitory action of NMDA on DKaiR1D (Li, 2016).
AMPA receptors were initially identified by and named in response to their activation by quisqualic acid, a glutamate bioisostere that is nonselective and which activates all of the major vertebrate iGluR subtypes, in addition to acting as a potent agonist for G protein-coupled glutamate receptors. Prior to the cloning of GluA1–4 subunits, the so-called quisqualate receptors were renamed AMPA receptors, following the synthesis of AMPA and the discovery that it was a highly selective agonist, without activity at kainate, NMDA, or G protein-coupled glutamate receptors. These serendipitous events in the history of the development of selective ligands for iGluR subtypes were strongly reinforced when a large family of vertebrate iGluR subunits were cloned, and it was discovered that these encoded discrete families of iGluR subtypes, each with high sequence identity, the ligand-binding properties of which corresponded to the familiar AMPA, kainate, and NMDA receptor subtypes. The current experiments reveal an unexpected breakdown of the classification scheme for Drosophila and most likely other insect species iGluRs (Li, 2016).
With the plethora of genetic tools and advanced connectome analyses, Drosophila has emerged as a key model organism for studying the circuit basis of behavior. It is now evident that like vertebrates, glutamatergic synapses are abundantly utilized in fly CNS circuits. Functional and structural analyses revealed that Drosophila iGluRs have agonist and antagonist selectivity very different from those of vertebrates, indicating that sequence and structural homology does not confer conserved pharmacological properties. However, the unique pharmacology of Drosophila iGluRs reported in this study has proven of use to reveal the role of KaiR1D in presynaptic homeostasis. It is envisioned that appropriate use of pharmacological tools in combination with powerful fly genetics will greatly aid studies of complex neural circuits in Drosophila (Li, 2016).
GABAergic signaling is the cornerstone for fast synaptic inhibition of neural signaling in arthropods and mammals and is the molecular target for insecticides and pharmaceuticals, respectively. The K+-Cl- cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular Cl- concentration, yet the fundamental physiology, comparative physiology, and toxicological relevance of insect KCC is understudied. Considering this, electrophysiological, genetic, and pharmacological methods were employed to characterize the physiological underpinnings of KCC function to the Drosophila CNS. The data show that genetic ablation or pharmacological inhibition of KCC results in an increased spike discharge frequency and significantly (P<0.05) reduces the CNS sensitivity to gamma-aminobutyric acid (GABA). Further, simultaneous inhibition of KCC and ligand-gated chloride channel (LGCC) complex results in a significant (P<0.001) increase in CNS spontaneous activity over baseline firing rates that, taken together, supports functional coupling of KCC to LGCC function. Interestingly, 75% reduction in KCC mRNA did not alter basal neurotransmission levels indicating that only a fraction of the KCC population is required to maintain the Cl- ionic gradient when at rest, but prolonged synaptic activity increases the threshold for GABA-mediated inhibition and reduces nerve sensitivity to GABA. These data expand current knowledge regarding the physiological role of KCC in a model insect and provides the necessary foundation to develop KCC as a novel biochemical target of insecticides as well as complements existing research to provide a holistic understanding of the plasticity in mammalian health and disease (Chen, 2019).
Transmembrane protein (TMEM16) family members play numerous important physiological roles, ranging from controlling membrane excitability and secretion to mediating blood coagulation and viral infection. These diverse functions are largely due to their distinct biophysical properties. Mammalian TMEM16A and TMEM16B are Ca(2+)-activated Cl(-) channels (CaCCs), whereas mammalian TMEM16F, fungal afTMEM16, and nhTMEM are moonlighting (multifunctional) proteins with both Ca(2+)-activated phospholipid scramblase (CaPLSase) and Ca(2+)-activated, nonselective ion channel (CAN) activities. To further understand the biological functions of the enigmatic TMEM proteins in different organisms, this study combined an improved annexin V-based CaPLSase-imaging assay with inside-out patch clamp technique to thoroughly characterized Subdued, a Drosophila TMEM ortholog. Subdued is also a moonlighting transport protein with both CAN and CaPLSase activities. Using a TMEM16F-deficient HEK293T cell line to avoid strong interference from endogenous CaPLSases, this functional characterization and mutagenesis studies revealed that Subdued is a bona fide CaPLSase. The finding that Subdued is a moonlighting TMEM expands understanding of the molecular mechanisms of TMEM proteins and their evolution and physiology in both Drosophila and humans (Le, 2019).
The ground-breaking discoveries of TMEM16A and TMEM16B as the long-sought CaCCs advanced the understanding of a novel membrane protein superfamily that includes the TMEM family and its closely related OSCA, TMEM and TMC membrane protein families. TMEM proteins have been found in fungi, amoeboids, insects and vertebrates. The unexpected findings of mammalian TMEM16F as a moonlighting protein, a special type of proteins that can perform two or more distinct functions without gene fusions, multiple RNA splice variants or multiple proteolytic fragments, advanced understanding of the enigmatic TMEM family (10-12,19,20). Serving as a bona fide CaPLSase and a small-conductance CAN (SCAN) channel, TMEM16F has evolved the capability to passively transport phospholipids and ions, two structurally distinct classes of permeants, down their chemical gradients (Le, 2019).
Upon Ca2+ binding, TMEM16FCaPLSase mediates the rapid flip-flopping of phospholipids across cell membranes and thus dissipates the asymmetric distribution of membrane phospholipids. During platelet activation, TMEM16F-CaPLSase-induced phosphatidylserine (PS) externalization is essential for prothrombinase assembly, subsequent thrombin generation and blood coagulation. Consistent with its importance in blood coagulation, both the Scott syndrome patients who carried TMEM16F loss-of-function mutations and TMEM16F deficient mice exhibited prolonged bleeding phenotype. Despite the known physiological function of TMEM16F-CaPLSase in blood coagulation, it is unclear whether and how TMEM16F's ion channel activity can participate in this process (Le, 2019).
Recent structural and functional studies elegantly revealed that the fungal nhTMEM16, afTMEM and mammalian TMEM16E were also moonlighting proteins with CaPLSase and channel activities. Interestingly, the mammalian TMEM16A and TMEM16B CaCCs only displayed ion channel activities, while an amoebozoa TMEM homolog from Dictyostelium discoideum only showed CaPLSase activity when heterologously expressed in HEK cells. In order to understand the biological functions of TMEM moonlighting, there is an urgent need to have an in-depth understanding of TMEM evolution and function in different kingdoms ranging from Protozoa, Fungi to Animalia (Le, 2019).
TMEM moonlighting proteins have not been identified thus far in insects, despite a recent study that clearly demonstrated the physiological importance of CaPLSase in the degeneration of Drosophila sensory neurons (Sapar, 2018). However, the molecular identity of the Drosophila CaPLSase responsible for the observed scramblase activities remains elusive. Among the five Drosophila TMEM homologs, Subdued is the only protein that has been thoroughly characterized using electrophysiological tools (Wong, 2013). When heterologously expressed in HEK293T cells, whole-cell patch clamp recordings suggested that Subdued was a CaCC. Interestingly, Subdued-deficient Drosophila exhibited severe defects in host defense when challenged with the pathogenic bacterium Serratia marcescens. It remains, however, unclear how Subdued CaCC function is involved in Drosophila's immunity (Le, 2019).
Combining an improved Annexin V-based CaPLSase imaging assay with inside-out patch clamping technique, this study has discovered that Subdued is also a moonlighting TMEM16 protein in Drosophila. Notably, it was also found that Subdued harbors biophysical features that strikingly resembled those of the mammalian TMEM16F, which has been unambiguously shown to function as a CaPLSase and a CAN channel. These results thus support the notion that TMEM moonlighting could be an ancient feature of TMEM family, which is conserved in fungi, insects and vertebrates. These study provided new insights into understanding the evolution of TMEM family, the molecular mechanisms of their ion and phospholipid permeation, as well as TMEM physiological functions in Drosophila (Le, 2019).
Protein moonlighting as both ion channels and phospholipid scramblases has been observed in mammal and fungal TMEM proteins. By using patch clamp electrophysiology and an improved phospholipid scrambling assay, these studies reveal that Drosophila Subdued, an insect TMEM16, is also a moonlighting protein that can serve as both a CAN channel and a CaPLSase (Le, 2019).
In this study, it was also shown that the widely used HEK293T cell line had endogenous TMEM16F expression and strong CaPLSase activity. The endogenous CaPLSase activity can interfere with characterization of exogenous TMEM CaPLSases and complicate subsequent interpretation. To circumvent this complication, CRISPR-Cas method was applied to generate a TMEM16F KO HEK293T cell line, which lacks endogenous CaPLSase activity and thereby can serve as an ideal heterologous expression system to characterize CaPLSase activities. When Subdued was heterologously expressed in this KO cell line, robust Subdued-mediated CaPLSase activity was observed. Disrupting a key conserved residue at the extracellular entrance of Subdued abolished its CaPLSase activity. In addition, when one of the conserved Ca2+-binding residues was replaced with a positively charged Arg residue, the mutant Subdued failed to scramble phospholipids\. Collectively, these data show that Subdued is a bona fide CaPLSase (Le, 2019).
Inside-out excised patch clamp recordings demonstrated that Subdued is a CAN channel with higher cation permeability than chloride (PNa/PCl = 5.83) in μM intracellular Ca2+. This conclusion stands in stark contrast to a previous study, which reported that Subdued functioned as a CaCC (PNa/PCl = 0.16) based on whole-cell patch recordings (Wong, 2013). We postulate that this discrepancy might be derived from the inherent differences between the two patch clamp configurations. First, infusion of pipette solution with high micromolar Ca2+ into cytosol could disrupt intracellular environment, which might subsequently alter channel activity. In the case of Subdued, channel current run-up was observed in whole-cell recording (Wong, 2013). When whole-cell recording was used to measure TMEM16F current, a to 15-minute delay of channel activation has been frequently observed after membrane break-in. Under inside-out configuration, both Subdued and TMEM16F current can be immediately recorded after membrane excision. Without the long delay to obtain stable current, the reversal potential measured using inside-out configuration may reflect the intrinsic channel selectivity. Second, whole-cell patch clamp may suffer from larger leak current during recording, especially when infusing with high micromolar Ca2+ into the cytosol. The potential leak current could confound the reversible potential measurement. Third, measuring the reversal potential requires exchanging solutions with drastically different ionic concentrations. Whole-cell recording usually requires whole-chamber solution exchange, which can induce large liquid junction potential to complicate reversal potential measurement. In the current inside-out patch clamp experiments, a pressurized focal perfusion system was used to achieve rapid solution exchange directly to the excised patch membrane. As this process is fast and only requires a small volume of solution, the impact of liquid junction potential is negligible (Le, 2019).
This study also found that Subdued ion permeability resembles that of the mammalian TMEM16F-SCAN. Similar to TMEM16F-SCAN (Yang, 2012), common CaCC blockers such as niflumic acid (NFA), flufenamic acid (FFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid
(NPPB) cannot block Subdued current (Wong, 2013), further supporting that Subdued channel is different from TMEM16A-CaCC. Interestingly, the fungal afTMEM and nhTMEM channels also exhibit substantial cation permeability. The non-selective nature of the moonlighting TMEM proteins towards phospholipids and ions suggests that their ancestors may have experienced low selective pressure during evolution, so that they could mediate simultaneous permeation of different ions and phospholipids without expanding the genome size. Interestingly, TMEM16A and TMEM16B are more selective to anions and lack CaPLSase function. It is hoped that the current findings can shine new lights on understanding TMEM evolution and molecular mechanisms for substrate selectivity.
This study also provides new insights into understanding the physiological functions of TMEM moonlighting proteins. Previous studies have shown that Subdued knocked-out or knocked-down Drosophila strains harbored defects in their host defense and exhibited lethality upon ingestion of the pathogenic bacteria S. marcescens (Wong, 2013; Cronin, 2009). It is thus far not clear whether Subdued's CaPLSase function and/or ion channel function play a major role in participating host defense. Interestingly, the moonlighting protein TMEM16F has also been reported to express in immune cells and play an important role in immune responses. Considering the fact that channel activation of both Subdued and TMEM CAN current requires both high Ca2+ and membrane depolarization, both of which conditions are unlikely to be achieved under physiological conditions, it is likely that their CaPLSase functions might play the major role in immunity. A recent study suggested that PS externalization induced by overexpressing mammalian TMEM16F- CaPLSase played an important role in controlling neurite degeneration in Drosophila sensory neurons (Sapar, 2018). The current finding that Subdued as a bona fide CaPLSase might help identify the CaPLSase that is responsible for Drosophila neuronal degeneration (Le, 2019).
Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. This study combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. A reduction was observed in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, these results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation (Hu, 2019).
In almost all animals, mature oocytes are arrested in meiosis at the end of oogenesis and require an external trigger to be activated and transition to start embryogenesis. This 'egg activation' involves multiple events that transition eggs to embryogenesis, including meiosis resumption and completion, maternal protein modification and/or degradation, maternal mRNA degradation or translation, and egg envelope changes (Hu, 2019).
Triggers of egg activation vary across species. In vertebrate and some invertebrate species, fertilization triggers egg activation. However, changes in pH, ionic environment, or mechanical pressure can also trigger egg activation in other invertebrate species. A conserved response to these triggers is a rise of intracellular free Ca2+ levels in the oocyte. This calcium rise is due to influx of external calcium and/or release from internal storage, depending on the organism. The elevated Ca2+ concentration is thought to activate Ca2+- dependent kinases and/or phosphatases, which in turn change the phospho-proteome of the activated egg, initiating egg activation events (Hu, 2019).
Drosophila eggs activate independent of fertilization and the trigger is mechanical pressure. When mature oocytes exit the ovary and enter the lateral oviduct, they experience mechanical pressure from reproductive tract tract. As the oocytes swell due to the influx of oviductal fluid, their envelopes become taut. Drosophila oocytes can be activated in vitro by incubation in a hypotonic buffer, although some egg activation events do not proceed completely normally in vitro. Intracellular calcium levels rise in oocytes occur egg activation, as observed with the calcium sensor GCaMP. This calcium rise takes the form of a wave that starts at the oocyte pole(s) and traverses the entire oocyte. Initiation of this calcium wave requires influx of external Ca2+, as chelating external Ca2+ in in vitro egg activation assays blocks the calcium wave and egg activation. Propagation of the calcium wave relies on the release of internal Ca2+ stores, likely through an Inositol 1,4,5-trisphosphate (IP3) mediated pathway, as knocking down the endoplasmic reticulum (ER) calcium channel IP3 receptor (IP3R) prevents propagation of the calcium wave (Hu, 2019).
How mechanical forces trigger calcium entry during Drosophila egg activation was unknown. However, the lack of initiation of a calcium wave in the presence of Gd3+, an inhibitor of mechanosensitive ion channels, and N-(p-Amylcinnamoyl) anthranilic acid (ACA), an inhibitor of TRP-family ion channels, suggested that TRP family ion channels are likely involved. Further supporting this idea is the recent discovery that a TRP family channel, TRPM7, is needed for calcium influx that leads to calcium oscillations in activating mouse eggs. The Drosophila genome encodes 13 TRP family channels, but according to RNAseq data, only 3 (Painless, Trpm, and Trpml) are expressed in the ovary. This study used specific inhibitors, existing mutants, germline- specific RNAi knockdown, and new knockouts that were created with CRISPR/Cas9 to screen these 3 candidates for their roles in the initiation of the calcium wave. Trpm, the single Drosophila ortholog of mouse TRPM7, was shown to mediate the calcium wave initiation, whereas the other two TRP channels are not necessary to initiate the calcium wave (Hu, 2019).
Calcium wave phenotypes are normal in oocytes from pain or trpml null mutants. However, the frequency of the calcium wave is diminished in wildtype oocytes in the presence of Trpm inhibitors and in oocytes from trpm germline knockdown or knockout mutants. These results consistently indicated that Trpm mediates the calcium influx that initiates the calcium wave during Drosophila egg activation. trpm germline knockout females also displayed significantly decreased egg hatchability, due to defects after cell cycle resumption. The reduced hatchability suggested that maternal trpm function or the calcium wave is required for further embryogenesis after egg activation (Hu, 2019).
TRP family ion channels are non-selective and respond to a wide array of environmental stimuli. Drosophila Trpm has been reported to play multiple roles throughout larval development, including maintaining Mg2+ and Zn2+ homeostasis (Georgiev, 2010; Hofmann, 2010), and sensing noxious cold in larval Class III md neurons (Turner, 2016). However, the role of Trpm in reproduction had not been investigated because of the pupal lethality of trpm null mutants. In this study germline specific RNAi knockdown and CRISPR/Cas9 mediated knockout revealed three novel functions of Drosophila Trpm: supporting early oogenesis, mediating influx of environmental calcium to initiate the calcium wave during egg activation, and maternally supporting embryonic development after egg activation (Hu, 2019).
A previous study suggested that calcium influx during Drosophila egg activation is mediated through mechanosensitive ion channels (Homer, 2008). Both Drosophila Trpm and its mouse ortholog TRPM7 are reported to be constitutively active and permeable to a wide range of divalent cations (Georgiev, 2010). Mouse TRPM7 is known to respond to mechanical pressure, but further study will be needed to determine whether Drosophila Trpm is similarly responsive to mechanical triggers, such as those that occur during ovulation (Hu, 2019).
Germline knockout of trpm significantly reduced the frequency of observing calcium waves in in vitro egg activation assays and egg hatchability. However, this reduced egg hatchability was not due to failure of cell cycle resumption during egg activation. There are two possible explanations for the reduced egg hatchability of trpm germline knockout females (Hu, 2019).
First, it is possible that trpm plays a maternal role, independent of its role in initiating the calcium wave, such that lack of maternally deposited Trpm proteins leads to defects during embryogenesis. In mouse, TRPM7 is also required for normal early embryonic development, apart from its role in calcium oscillations. Inhibition of TRPM7 function impairs pre-implantation embryo development and slows progression to the blastocyst stage. Drosophila trpm mutant lethality had been reported to occur during the pupal stage. However, those homozygous mutants were offspring of heterozygous mothers, and thus did not lack maternal Trpm function. Germline specific depletion of trpm reveals a maternal role for Trpm in embryogenesis (Hu, 2019).
Alternatively, or in addition, it is possible that oocytes lacking Trpm do not take up sufficient Ca2+ from the environment to form a calcium wave, but that at least some events of egg activation can occur despite this. In mouse, an initial calcium rise is induced by sperm-delivered PLCζ via the IP3 pathway. Yet although sperm from PLCζ null males fails to trigger normal calcium oscillations, some eggs fertilized by those sperm develop. Multiple oscillations following fertilization require influx of external calcium, mediated by TRPM7 and CaV3.2 (Bernhardt, 2018). Even though these oscillations were reported to be needed for multiple post-fertilization events, some TRPM7 and CaV3.2 double-knockout embryos still develop, albeit not completely normally. Together, these data suggest that egg activation can still occur in mouse with diminished intracellular calcium rises, analogous to what is seen in Drosophila in the absence of maternal Trpm function (Hu, 2019).
Insufficient influx of calcium in the absence of Trpm function could disrupt later (but maternally- dependent) embryogenesis. The oocyte-to-embryo transition involves multiple events. In mouse
egg activation, these events take place sequentially as calcium oscillations progress, with developmental progression associated with more oscillations and more total calcium signal. Some of the events start after a certain number of oscillations but require additional oscillations to complete. It is possible that mechanisms critical for Drosophila embryo development also depend on reaching a precise level of calcium. A low-level calcium rise might be sufficient to trigger some egg activation mechanisms such as vitelline membrane crosslinking and cell cycle resumption, but high-levels of calcium may be required for further progression (Hu, 2019).
Given the importance of calcium in egg activation, it was surprising that although trpm knockout eggs lacked a calcium wave in vitro, in vivo such eggs could progress in cell cycles and even, sometimes, hatch. There may be insufficient calcium influx in the absence of Trpm for full and efficient development, but some egg activation events may still occur. Alternatively, it is possible that redundant mechanisms permit a sufficient calcium-level increase without producing a detectable wave form. Despite being able to trigger a series of egg activation events including meiosis resumption and protein translation, osmotic pressure during in vitro activation may have different properties from mechanical pressure exerted on mature oocytes during ovulation. The latter might allow opening of other calcium channels to initiate a normal calcium rise and complete egg activation. Two channels, TRPM7 and Cav3.2, are needed for the calcium oscillations following mouse fertilization, but the Drosophila ortholog of mouse Cav3.2, Ca-α1T, is not detectably expressed in fly ovaries. Other unknown channels might play this redundant role in vivo. In this light it is noted that levels of basal GCaMP fluorescence varied among oocytes incubated in IB that were inhibited from forming a wave, suggesting the possibility of a calcium increase by a redundant mechanism (Hu, 2019).
It was intriguing that Drosophila Trpm is essential for the calcium rise at egg activation, and that its mouse ortholog, TRPM7, was recently reported to be required (along with CaV3.2) for the calcium influx needed for post-fertilization calcium oscillations that are in turn required for egg activation events. This apparent conservation in mechanisms in egg activation involving orthologous Trpm channels in a protostome (Drosophila) and a deuterostome (mouse) prompts asking whether Trpm-mediated calcium influx is a very ancient and basal aspect of egg activation, with other more variable aspects such as sperm-triggered calcium rises being more derived, if better known, features. It is interesting in this light that a sperm-delivered TRP channel (TRP-3) has also been reported to mediate calcium influx and a calcium rise in another protostome, C. elegans (Hu, 2019).
In cells, organs and whole organisms, nutrient sensing is key to maintaining homeostasis and adapting to a fluctuating environment. In many animals, nutrient sensors are found within the enteroendocrine cells of the digestive system; however, less is known about nutrient sensing in their cellular siblings, the absorptive enterocytes. This study used a genetic screen in Drosophila melanogaster to identify Hodor, an ionotropic receptor in enterocytes that sustains larval development, particularly in nutrient-scarce conditions. Experiments in Xenopus oocytes and flies indicate that Hodor is a pH-sensitive, zinc-gated chloride channel that mediates a previously unrecognized dietary preference for zinc. Hodor controls systemic growth from a subset of enterocytes-interstitial cells-by promoting food intake and insulin/IGF signalling. Although Hodor sustains gut luminal acidity and restrains microbial loads, its effect on systemic growth results from the modulation of Tor signalling and lysosomal homeostasis within interstitial cells. Hodor-like genes are insect-specific, and may represent targets for the control of disease vectors. Indeed, CRISPR-Cas9 genome editing revealed that the single hodor orthologue in Anopheles gambiae is an essential gene. These findings highlight the need to consider the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis (Redhai, 2020).
To investigate nutrient sensing in enterocytes, 111 putative nutrient sensors in D. melanogaster were selected on the basis of their intestinal expression and their predicted structure or function. Using two enterocyte-specific driver lines, their expression was downregulated in midgut enterocytes throughout development under two dietary conditions, nutrient-rich and nutrient-poor; it was reasoned that dysregulation of nutrient-sensing mechanisms may increase or reduce the normal period of larval growth, and might do so in a diet-dependent manner. Enterocyte-specific knockdown of the gene CG11340, also referred to as pHCl-22, resulted in developmental delay. This delay was exacerbated, and was accompanied by significantly reduced larval viability, under nutrient-poor conditions; these phenotypes were confirmed using a second RNAi transgene and a new CG11340 mutant. In the tradition of naming Drosophila genes according to their loss-of-function phenotype, CG11340 was named 'hodor', an acronym for 'hold on, don't rush', in reference to the developmental delay (Redhai, 2020).
A transcriptional reporter revealed that Hodor was expressed in the intestine. A new antibody revealed that Hodor expression was confined to enterocytes in two midgut portions that are known to store metals: the copper cell region and the iron cell region. Within the copper cell region, Hodor was expressed only in so-called interstitial cells. hodor-Gal4 was also present in the interstitial cells of the copper cell region; however, in the experimental conditions used in this study and in contrast to published results, it was not detected in the iron cell region. Apart from the intestine, Hodor was found only in principal cells of the excretory Malpighian tubules. To identify the cells from which Hodor controls systemic growth, region- or cell-type-specific downregulation and rescue experiments were conducted. Only fly lines in which hodor was downregulated in interstitial cells showed slowed larval development. This developmental delay persisted when hodor knockdown was induced post-embryonically during larval growth, and was rescued only in fly lines in which hodor expression was re-instated in cell types that included interstitial cells. The fat body (analogous to liver and adipose tissue) has long been known to couple nutrient availability with developmental rate; however, recent studies have revealed contributions from the intestine, particularly in nutrient-poor conditions. The current findings confirm a role for the intestine in coupling nutrient availability with larval growth, and further implicate a subpopulation of enterocytes-interstitial cells-as important mediators. Interstitial cells were described decades ago in blowfly, but had remained relatively uncharacterized since; their name refers only to their position, interspersed among the acid-secreting copper cells that control microbiota loads (Redhai, 2020).
This study established that the lethality of hodor mutation or knockdown was apparent only during the larval period. The development of hodor mutants was slower throughout larval life, and surviving mutants attained normal pupal and adult sizes. Consistent with previous findings, hodor mutation or knockdown was found to reduce luminal acidity in the copper cell region, suggesting a role specifically for interstitial cells in this process. hodor mutants also had increased gut bacterial titres, which is consistent with the observed functional defects in the copper cell region. Enlarged volumes of both the lumen of the copper cell region and the interstitial cells were also apparent after 1-3 days of (delayed) larval development; ultrastructurally, this was apparent in interstitial cells as a reduction in the complexity of their characteristic basal infoldings. This study was, however, able to rule out all of these defects as reasons for the developmental delay (Redhai, 2020).
During the course of these experiments, it was observed that hodor mutant larvae were more translucent than control larvae. This was suggestive of peripheral lipid depletion, which was confirmed by quantifying and staining for triacylglycerides. Reduced lipid stores did not result from disrupted enterocyte integrity: the intestinal barrier of mutants was intact, both anatomically and functionally. It was observed that hodor mutants had less food in their intestines and accumulated insulin-like peptide Ilp2 in their brains (nutrient-dependent Ilp2 secretion promotes larval development; its accumulation in the brain is commonly interpreted as peptide retention in the absence of transcriptional changes). Consistent with reduced systemic insulin signalling, hodor mutant larval extracts had reduced levels of phospho-Akt and phospho-S6 kinase. As these are all indicators of starvation, food intake was quantified, and it was observed to be reduced in both hodor mutant larvae and in hodor knockdowns targeting interstitial cells. Reduced food intake was apparent soon after hatching and persisted throughout larval development. Ectopic expression of Ilp2-which rescues developmental delay in larvae that lack insulin-like peptides-in hodor mutants partially rescued their developmental delay, but did not increase their food intake. An 'instructive' link between intestinal Hodor and food intake was further suggested by the overexpression of hodor in otherwise wild-type enterocytes; this resulted in larvae that ate more and developed at a normal rate, but had increased lipid stores. Therefore, Hodor controls larval growth from a subset of enterocytes by promoting food intake and systemic insulin signalling. In its absence, larvae fail to eat sufficiently to proceed through development at the normal rate and are leaner. When present in excess, Hodor causes larvae to eat more and accumulate the energy surplus as fat (Redhai, 2020).
In fly adipose tissue, amino acid availability activates Tor signalling to promote systemic growth. This study therefore combined hodor knockout or knockdown with genetic manipulations to alter Tor signalling. In flies with reduced or absent Hodor function, decreasing or increasing Tor signalling in hodor-expressing cells exacerbated or rescued the developmental delay, respectively. The reduced food intake of hodor mutants was also significantly rescued by activation of Tor signalling in hodor-expressing cells. Genetic targeting of Rag GTPases or the Gator1 complex in these cells failed to affect the developmental delay of hodor mutants, which could suggest non-canonical regulation of Tor signalling in Hodor-expressing cells. The systemic effects of Hodor on food intake and larval growth are therefore modulated by Tor signalling within Hodor-expressing interstitial cells (Redhai, 2020).
Hodor belongs to the (typically neuronal) Cys-loop subfamily of ligand-gated ion channels, and is predicted to be a neurotransmitter-gated anion channel. It is known to show activity in response to alkaline conditions in Xenopus oocytes, but the acidic pH of the copper cell region prompted a search for additional ligands. Although alkaline pH-induced Hodor activity was confirmed in oocyte expression systems, Hodor did not respond to typical Cys-loop receptor ligands such as neurotransmitters or amino acids. Instead, the screen identified zinc as an unanticipated ligand, which elicited a strong dose-dependent response only in Hodor-expressing oocytes; this response to zinc showed peak current amplitude values much greater than those observed in response to pH or to other metals such as iron or copper. Force-field-based structural stability and binding affinity calculations identified the amino acid pair E255 and E296 as a potential binding site for the divalent zinc ion. Mutating these residues did not abrogate the zinc-elicited currents, but did result in currents with faster rise time and deactivation kinetics, which supports the idea that zinc is a relevant Hodor ligand. On the basis of its sequence and conductance properties, Hodor has been proposed to transport chloride (Feingold, 2016; Remnant, 2016), and the zinc-elicited currents that were observed in oocytes had a reversal potential that is consistent with chloride selectivity. In vivo experiments in flies showed that supplementation of a low-yeast diet with zinc led to a reduction of chloride levels in interstitial cells, whereas hodor mutation increased chloride levels. Thus, Hodor is a pH-modulated, zinc-gated chloride channel (Redhai, 2020).
Attempts were made to establish the relevance of zinc binding in vivo. Zinc enrichment is observed in both the copper and iron cell regions of the larval gut, revealing an unrecognized role for these Hodor-expressing regions in zinc handling. Mutation of hodor failed to affect this zinc accumulation, although dietary yeast levels did, which is consistent with a role for Hodor in sensing rather than transporting zinc. (Notably, the white mutation-which is frequently used in the genetic background of Drosophila experiments-results in a small but significant reduction in both intestinal zinc accumulation and larval growth rate, although the status of the w gene neither exacerbated nor masked the more substantial, hodor-induced developmental delay. Furthermore, larvae that were fed a low-yeast diet ate significantly more when the diet was supplemented with zinc; this effect was abrogated in hodor mutants. In a food choice experiment, control larvae developed a preference for zinc-supplemented food over time, which suggests that the preference develops after ingestion. Consistent with this idea, zinc preference was specifically abrogated in hodor mutants (their general ability to discriminate between other diets was confirmed. Thus, zinc sensing by Hodor is physiologically relevant in vivo. Metals such as zinc are primarily provided by yeasts in nature; Hodor may be one of several sensors used to direct larvae to nutrient-rich food sources (Redhai, 2020).
The subcellular localization of Hodor suggests that it may normally maintain low cytoplasmic chloride concentrations by transporting it out of the interstitial cells and/or into their lysosomes. In accordance with this, and consistent with its putative lysosomal localization signals, Hodor was specifically enriched in apical compartments containing late endosome or lysosomal markers, as well as decorating the brush border of interstitial cells. The presence of Hodor in a subpopulation of lysosomes was of interest, because chloride transport across lysosomal membranes often sustains the activity of the proton-pumping vacuolar-type ATPase (V-ATPase) that maintains lysosomal acidity and Tor activation on the lysosome. To explore a role for Hodor in enabling Tor signalling, whether the absence of hodor induced autophagy-a hallmark of reduced Tor signalling, was tested. First, the induction of common autophagy markers in interstitial cells after genetic interference with the V-ATPase complex, which is known to promote autophagy by reducing lysosomal acidity and Tor signalling, was confirmed. Similar to reduced V-ATPase function, loss of hodor increased autophagy in interstitial cells. Expression of the dual autophagosome and autolysosome reporter UAS-GFP-mCherry-Atg8a in the intestinal cells of hodor mutants confirmed the induction of autophagy, and revealed two additional features. First, the acidification of autophagic compartments was defective in hodor mutants. Second, the increased autophagy and defective acidification observed in hodor mutants were particularly prominent in the two Hodor-expressing intestinal regions (the copper cell region and the iron cell region), consistent with cell-intrinsic roles for Hodor in these processes. Additional support for the roles of lysosomal function and Tor signalling in controlling whole-body growth from interstitial cells was provided by the finding that most V-ATPase subunits were transcriptionally enriched in the copper cell region. Functionally, the downregulation of V-ATPase subunits specifically in Hodor-expressing cells-and not in other subsets of enterocytes, such as those targeted by R2R4-Gal4-led to developmental delay and reduced food intake, phenotypes comparable to those observed as a result of hodor downregulation. Hence, although the directionality of zinc sensing and chloride transport in interstitial cells remains to be established, the data are consistent with roles for brush-border Hodor in transporting chloride out of interstitial cells-thus maintaining osmolarity and water balance. Lysosomal Hodor may transport chloride into the lysosome to sustain V-ATPase function, lysosomal acidification and TOR signalling, pointing to new links between lysosomal homeostasis in specialized intestinal cells, food intake and systemic growth. Nutrients such as amino acids are important regulators of Tor signalling. The genetic data are consistent with novel input from metals and/or micronutrients into Tor signalling. The nutrient-dependent zinc accumulation in lysosomal organelles-recently described in mammalian cells and nematode worms-suggests that links between zinc, lysosomes and Tor may be of broader importance. Two attractive cell types in which to explore such links are the Paneth cells of the mammalian intestine, which accumulate zinc and regulate intestinal immunity and stem cell homeostasis, and the 'lysosome-rich enterocytes' that have recently been described in fish and mice, which have roles in protein absorption (Redhai, 2020).
An extensive reconstruction of the hodor family tree supported the presence of a single member of the family in the ancestor of insects. Because Hodor-like proteins are present only in insects, they may prove to be highly specific targets for the chemical control of disease vectors, particularly given that mosquito genomes contain a single gene rather than the three paralogues that are found in most flies. To test this idea, CRISPR-Cas9 genome editing was used to generate a mutant that lacks the single hodor-like gene (AGAP009616) in the malaria vector Anopheles gambiae. This gene is also expressed in the digestive tract-specifically in the midgut-and in Malphighian tubules. Three independent deletion alleles revealed that AGAP009616 function is essential for the viability of A. gambiae. A target that is expressed in the intestine, such as Hodor, is particularly attractive for vector control as it may circumvent accessibility issues and could be directly targeted using ingestible drugs such as those applied to larval breeding sites (Redhai, 2020).
Metals have received little attention in the contexts of development or whole-body physiology, and are often regarded as passive 'building blocks'. By revealing the roles of a metal sensor in food intake and growth control, these findings highlight the importance of investigating the instructive contributions of metals-and, more generally, micronutrients-to energy homeostasis. These mechanisms could prove to be useful in insect vector control (Redhai, 2020).
Non-steroidal anti-inflammatory drugs (NSAIDs) are used for relieving pain and inflammation accompanying numerous disease states. The primary therapeutic mechanism of these widely used drugs is the inhibition of cyclooxygenase 1 and 2 (COX1, 2) enzymes that catalyze the conversion of arachidonic acid into prostaglandins. Transient receptor potential melastatin 7 (TRPM7) cation channels are highly expressed in T lymphocytes and are inhibited by Mg(2+), acidic pH, and polyamines. This study reports a novel effect of naproxen, ibuprofen, salicylate, and acetylsalicylate on TRPM7. At concentrations of 3-30mM, they reversibly inhibited TRPM7 channel currents. By measuring intracellular pH with the ratiometric indicator BCECF, it was found that at 300μM to 30mM, these NSAIDs reversibly acidified the cytoplasm in a concentration-dependent manner, and it is proposed that TRPM7 channel inhibition is a consequence of cytosolic acidification, rather than direct. NSAID inhibition of TRPM7 channels was slow, voltage-independent, and displayed use-dependence, increasing in potency upon repeated drug applications. The extent of channel inhibition by salicylate strongly depended on cellular PI(4,5)P(2) levels, as revealed when this phospholipid was depleted with voltage-sensitive lipid phosphatase (VSP). Salicylate inhibited heterologously expressed wildtype TRPM7 channels but not the S1107R variant, which is insensitive to cytosolic pH, Mg(2+), and PI(4,5)P(2) depletion. NSAID-induced acidification was also observed in Schneider 2 cells from Drosophila, an organism that lacks orthologous COX genes, suggesting that this effect is unrelated to COX enzyme activity. A 24-h exposure to 300μM-10mM naproxen resulted in a concentration-dependent reduction in cell viability. In addition to TRPM7, the described NSAID effect would be expected to apply to other ion channels and transporters sensitive to intracellular pH (Chokshi, 2021).
Insect nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels mainly expressed in the central nervous system of insects. They are the directed targets of many insecticides, including neonicotinoids, which are the most widely used insecticides in the world. However, the development of resistance in pests and the negative impacts on bee pollinators affect the application of insecticides and have created a demand for alternatives. Thus, it is very important to understand the mode of action of these insecticides, which is not fully understood at the molecular level. This study systematically examined the susceptibility of ten Drosophila melanogaster nAChR subunit mutants to eleven insecticides acting on nAChRs. The results showed that there are several subtypes of nAChRs with distinct subunit compositions that are responsible for the toxicity of different insecticides. At least three of them are the major molecular targets of seven structurally similar neonicotinoids in vivo. Moreover, spinosyns may act exclusively on the α6 homomeric pentamers but not any other nAChRs. Behavioral assays using thermogenetic tools further confirmed the bioassay results and supported the idea that receptor activation rather than inhibition leads to the insecticidal effects of neonicotinoids. The present findings reveal native nAChR subunit interactions with various insecticides and have important implications for the management of resistance and the development of novel insecticides targeting these important ion channels (Lu, 2022).
Acid taste, evoked mainly by protons (H(+)), is a core taste modality for many organisms. The hedonic valence of acid taste is bidirectional: animals prefer slightly but avoid highly acidic foods. However, how animals discriminate low from high acidity remains poorly understood. To explore the taste perception of acid, the fruit fly was used as a model organism. Flies employ two competing taste sensory pathways to detect low and high acidity, and the relative degree of activation of each determines either attractive or aversive responses. Moreover, one member of the fly Otopetrin family, Otopetrin-like a (OtopLa), was established as a proton channel dedicated to the gustatory detection of acid. OtopLa defines a unique subset of gustatory receptor neurons and is selectively required for attractive rather than aversive taste responses. Loss of otopla causes flies to reject normally attractive low-acid foods. Therefore, the identification of OtopLa as a low-acid sensor firmly supports a competition model of acid taste sensation. Altogether, this study has discovered a binary acid-sensing mechanism that may be evolutionarily conserved between insects and mammals (Mi, 2021).
Sour taste, like sweet, bitter, salty, and umami tastes, represents a fundamental taste modality across many species ranging from insects to mammals. Typically, humans like slightly acidic foods such as lemon juice, which potentially indicates the presence of nutrients. In contrast, humans dislike highly acidic foods, which can cause digestive tract tissue injuries. The bivalent taste response to acid is also documented in rodents. Similar to mammals, the fruit fly, Drosophila melanogaster, prefers low levels of acid, which stimulate feeding and reproduction, and avoids high acid concentrations. Therefore, although flies and humans appear drastically different, the hedonic valence of their acid-taste response is similar: it can be either attractive or aversive, depending on the acid concentration of food. It is proposed that the bidirectional characteristic of acid perception constitutes an evolutionary fitness that enables animals to choose nutritious and reject unhealthy food sources. How do animals make this seemingly challenging decision? It is hypothesized that a taste-coding mechanism underlies the opposing feeding behavior triggered by low and high levels of acids. However, the molecular and cellular nature of the acid-taste coding has remained unclear (Mi, 2021).
Several lines of research demonstrate that type III taste receptor cells (TRCs) are responsible for acid sensing in mice. Nevertheless, the type III TRC population may be heterogeneous and contain different cell subtypes. Due to the lack of molecular markers and genetic tools to manipulate different subsets of type III TRCs, the question of how type III TRCs differentially respond to different concentrations of acid appears to be difficult to address in mammals. Moreover, other than eliciting taste sensation, acid also activates trigeminal nerves in the oral cavity of mammals, leading to burning or pain sensation. This side effect further confounds the investigation of sour-taste coding and sour-taste-triggered behavior in mammals. In contrast, flies exhibit much more pronounced and distinct taste responses to varying concentrations of acid than do mammals. Therefore, the fly serves as an excellent animal model to elucidate the taste coding of acid. This study reports that the fly mainly uses two different subsets of gustatory receptor neurons (GRNs) to selectively sense low or high concentrations of acids. The taste transduction pathways orchestrated by low- and high-acid GRNs antagonize each other, and the net behavioral response to a particular concentration of acid is predominantly determined by the relative activities of low- vs. high-acid GRNs (Mi, 2021).
Animals take advantage of highly diversified taste receptors and TRCs to detect varying taste substances, including sugar, salt, acid, and bitter compounds. In mammals, in contrast to the well-characterized sweet and bitter receptors, the molecular identity of sour-taste receptor had not been determined until a recent discovery showing that the Otopetrin (Otop) protein family functions as proton channels. In mice, one of the Otop family members, Otop1, is essential for sour-taste transduction. Despite this significant finding, the exact role played by Otop1 in discriminating low- from high-acid foods remained unclear. As the Otop family is fairly conserved between mammals and insects, it was of interest to see if the Otop family is also required for taste sensation of acids in Drosophila, given that no bona fide sour-taste receptors had been established in insects. One of the fly Otop orthologues, Otopetrin-like a (OtopLa), was shown to act as a proton channel and is selectively required for attractive taste sensation of acids in Drosophila. The fly OtopLa protein is localized at the tip of the GRN dendrite, the forefront site of taste sensory cells that is responsible for directly sensing tastant stimuli. Further, OtopLa defines a novel class of GRNs, which are largely distinct from other groups of GRNs responding to sugar, salt, or bitter tastants. Furthermore, genetic analysis showed that loss of otopetrin-like a (otopla) selectively abolishes the attractive acid-taste pathway, leaving the aversive pathway intact. Notably, the otopla mutant flies became abnormally averse to low concentrations of acid. Thus, this study provides strong genetic evidence to establish not only that the attractive and aversive taste pathways responsible for acid sensation exist but also that they are genetically segregated. Finally, by establishing OtopLa as a bona fide taste receptor for acid in flies, this work overturns the long-standing view that insects and mammals use fundamentally different gustatory receptors (Mi, 2021).
According to our behavioral assays, the wild-type fly displays opposing taste responses to low and high concentrations of acid: low concentrations are attractive, whereas high concentrations are aversive. Thus, the hedonic valence of acid taste is closely associated with the concentration of acid that the animals detect. The bidirectional valence of sour taste is reminiscent of salty taste, which is also dependent on salt concentrations. Given these findings, a key question arises as to how the animals discern low- from high-acid foods. Rlectrophysiology analyses of different groups of taste sensilla provide an important clue to this question. Flies were found to mainly use L- and S-type sensilla to perceive low and high concentrations of acid, respectively. The L-type sensilla mediate an attractive pathway, whereas the S-type sensilla operate an aversive pathway for the response to acids. It is postulated that acid-taste signals relayed by low- and high-acid GRNs antagonize each other in the brain, where feeding decisions are made. When the fly encounters low-acid foods, the attractive pathway mediated by low-acid GRNs dominates the aversive pathway mediated by the high-acid GRNs, driving the animal to choose the low-acid food. Conversely, when the animal encounters high-acid foods, the aversive pathway dominates the attractive pathway, leading to the avoidance of high-acid foods (Mi, 2021).
In support of this hypothesis, genetic analysis reveals that otopla is specifically required for the attractive rather than aversive acid-taste response. Further, we found that otopla is mostly expressed in the GRNs housed within the L-type rather than the S-type sensilla. In addition, recent work shows that a member of the ionotropic receptor (IR) family, Ir7a, is selectively required for the repulsive response to high concentrations of acetic acid in Drosophila. However, flies lacking Ir7a show normal attractive feeding responses to low concentrations of acetic acid. That study, combined with the present study on otopla, provides substantial evidence to support the model that the attractive and aversive pathways for acid sensation are segregated in the peripheral taste organ (Mi, 2021).
These findings lay the foundation for a more detailed analysis of the genetic program and neural circuit involved in sour-taste perception. In mammals, type III TRCs are mainly responsible for sour-taste sensation. Nevertheless, whether distinct subgroups of type III TRCs in the taste bud selectively respond to low or high acid remains an open question. Given the conservation of acid-taste sensation between flies and mammals, the acid-taste coding mechanism identified in the fly will inform the investigation of sour-taste coding in mammals, including humans (Mi, 2021).
Using multiple lines of evidence, these studies lead to the identification of OtopLa as a long-sought taste receptor for acid in Drosophila. First, genetic analyses show that OtopLa is both necessary and sufficient to orchestrate the attractive response to foods containing low concentrations of acid. In addition, loss of otopla has no effect on sweet, bitter, or salty tastes. Second, cell biological studies reveal that OtopLa is expressed in a group of GRNs different from sweet, bitter, and salty GRNs. Moreover, OtopLa proteins selectively reside in the distal portion of the dendrite, the forefront of the GRN responsible for detecting taste substances presented from the food environment. Last but not least, patch-clamp recordings reveal that OtopLa functions as a proton channel and can be directly activated by protons. Collectively, this work establishes OtopLa as a bona fide receptor that is dedicated to the attractive taste sensation of acids in Drosophila. Recent studies in mice show that the Otop1 proton channel is both necessary and sufficient for sour-taste transduction. In light of these discoveries in flies and mice, it is concluded that the Otop family is an evolutionarily conserved proton channel dedicated to taste the sensation of acids in both insects and mammals. Over the past two decades, various types of taste receptors, including sweet, bitter, and salty taste receptors, have been identified and functionally characterized in both invertebrates and vertebrates. Although insects and mammals exhibit a striking homology in taste responses, the molecular identities of their sweet, bitter, and salty taste receptors appear to be distantly related to each other. Consequently, there is a long-held view in the chemoreception field that taste receptors for insects and mammals are evolutionarily distant from each other. In this study, the discovery in the fly acid-taste sensation has overturned this notion. The Otop family represents the first class of taste receptors that is functionally conserved between insects and mammals. From an evolutionary perspective, it is proposed that Otop is a well-conserved proton channel family involved in acid-taste sensation throughout the animal kingdom. Thus, further research is needed to explore the gustatory role of the Otop family in other animal species, including humans (Mi, 2021).
otopla is necessary for the attractive taste sensation of both the strong acid HCl and the weak acids citric acid and malic acid. Psychophysical studies in human subjects report that weak acid usually tastes more sour than strong acid at the same pH40, implying that, in addition to protons, the undissociated weak acid molecules may also elicit sourness. These studies demonstrate that the proton channel OtopLa is broadly required for the taste sensation of both strong and weak acids. Therefore, it is proposed that the taste response to acids orchestrated by OtopLa mainly results from the gustatory stimuli of protons that are dissociated from either strong or weak acids. In addition, several members of the fly IR family are involved in taste responses to carbonation and acetic acid. As there has been no evidence showing that the IRs form a proton channel, the IRs are likely to be narrowly tuned to the specific structures of weak acids rather than to protons. Collectively, the fly may use different taste transduction pathways to perceive various acid molecules present in the environment (Mi, 2021).
In conclusion, given the significant conservation of taste receptors for acid between flies and mammals, the fly model will significantly advance understanding of the acid-taste sensation in other animals, including humans (Mi, 2021).
Bacterial septicemia is commonly induced by Gram-negative bacteria. The immune response is triggered in part by the secretion of bacterial endotoxin lipopolysaccharide (LPS). LPS induces the subsequent release of inflammatory cytokines which can result in pathological conditions. There is no known blocker to the receptors of LPS. The Drosophila larval muscle is an amendable model to rapidly screen various compounds that affect membrane potential and synaptic transmission such as LPS. LPS induces a rapid hyperpolarization in the body wall muscles and depolarization of motor neurons. These actions are blocked by the compound doxapram (10 mM), which is known to inhibit a subtype of the two-P-domain K+ channel (K2P channels). However, the K2P channel blocker PK-THPP had no effect on the Drosophila larval muscle at 1 and 10 mM. These channels are activated by chloroform, which also induces a rapid hyperpolarization of these muscles, but the channels are not blocked by doxapram. Likewise, chloroform does not block the depolarization induced by doxapram. LPS blocks the postsynaptic glutamate receptors on Drosophila muscle. Pre-exposure to doxapram reduces the LPS block of these ionotropic glutamate receptors. Given that the larval Drosophila body wall muscles are depolarized by doxapram and hyperpolarized by chloroform, they offer a model to begin pharmacological profiling of the K2P subtype channels with the potential of identifying blockers for the receptors to mitigate the actions of the Gram-negative endotoxin LPS (Cooper, 2022).
Individual sensory neurons can be tuned to many stimuli, each driving unique, stimulus-relevant behaviors, and the ability of multimodal nociceptor neurons to discriminate between potentially harmful and innocuous stimuli is broadly important for organismal survival. Moreover, disruptions in the capacity to differentiate between noxious and innocuous stimuli can result in neuropathic pain. Drosophila larval Class III (CIII) neurons are peripheral noxious cold nociceptors and innocuous touch mechanosensors; high levels of activation drive cold-evoked contraction (CT) behavior, while low levels of activation result in a suite of touch-associated behaviors. However, it is unknown what molecular factors underlie CIII multimodality. This study showed that the TMEM16/anoctamins subdued and This study has shown that CIII cold nociceptors make use of excitatory Cl- currents to selectively encode cold. A current working hypothesis in light of these findings is that cold-evoked, TRP-channel mediated Ca2+ currents activate Cl- channel (CaCCs), which due to differential expression of ncc69 and kcc, result in depolarizing Cl- currents, enhancing neural activation in response to cold. These results support a role for subdued, white walker, and ncc69 in selectively facilitating CIII-dependent cold nociception and not mechanosensation, thereby participating in mechanisms that allow CIII neurons to differentiate between sensory modalities. While these results provide strong evidence for Ca2+-dependent mechanisms in the rapid response of CIII neurons to cooling, these studies also suggest that additional Ca2+-independent mechanisms may also contribute to complex processes in these neurons that function in driving spiking activity, sensitization, and/or cold acclimation at colder temperaturespain (Himmel, 2023).
As subdued has been previously characterized as a CaCC, its role is consistent with the hypothesis outlined above. However, the evolution of subdued has been implicitly debated in the literature, with suggestion that it may be more closely related to ANO6. Phylogenetic analysis carried out for this study strongly evidences that subdued is part of the bilaterian ANO1/ANO2 subfamily of CaCCs. Moreover, the phylogeny suggests that insects have no direct ANO6 homologue, as the diversification of ANO3, ANO4, ANO5, ANO6, and ANO9 occurred after the protostome-deuterostome split. The role of subdued in cold nociception therefore may constitute functional homology in the bilaterian ANO1/ANO2 subfamily, as mammalian ANO1 has been shown to participate in nociception alongside mammalian TRP channels. However, the possibility of convergent evolution cannot yet be ruled due to the absence of evidence of function in other taxa (Himmel, 2023).
In contrast, white walker has not been demonstrated to function as, or be closely related to, CaCCs. Phylogeny evidences that White walker is part of the metazoan ANO8 subfamily; one important function of mammalian ANO8 is to tether the endoplasmic reticulum (ER) and plasma membrane (PM), thereby facilitating inter-membrane Ca2+ signaling. Therefore, a speculative hypothesis is that White walker likewise serves to couple the ER and PM, and that subsequently, ER-dependent Ca2+ signaling might promote the CIII cold response. In fact, a recent study (published contemporaneously with this study) has shown that ER-related Ca2+-induced Ca2+ release mechanisms are required for cold nociception (Patel et al., 2022). However, ANO8 has been shown to conduct Cl- heterologously (Tian, 2012), so Cl- channel function in Drosophila cannot be ruled out a priori. As white walker appears to be broadly expressed in neural tissues, white walker may function as a fundamental component of insect neural machinery and is therefore likely to be a gene of interest in future studies (Himmel, 2023).
In addition to the functions outlined above, anoctamins-including Subdued (Le, 2019)-are known to function as lipid scramblases. A plausible alternative hypothesis is therefore that Subdued and/or White walker function as lipid scramblases as part of unidentified signaling cascades critical to noxious cold transduction (Himmel, 2023).
The results of ncc69 knockdown behavior, Cl--channel optogenetics, and Cl- electrophysiology experiments are consistent with the hypothesis that CIII neurons make use of atypical excitatory Cl- currents. However, no effect on cold-evoked CIII activity was observed. in response to ncc69 knockdown. It may be the case that this knockdown only affects electrical activity at very noxious temperatures; the inability to detect deficiencies in cold-evoked neural activity may therefore be due to limitations in the electrophysiology prep, which limit the ability to cool below 10°C. These results are still curious; however, as subdued and white walker knockdowns result in electrophysiological defects at less-noxious (10°C) and innocuous (15°C) temperature drops; despite this discrepancy, these electrophysiological differences do not correlate with behavioral differences at 10°C and decreased activity thus may not be behaviorally relevant at this temperature. Moreover, although there is substantial Bayesian evidence of an effect on % strong CT under kcc overexpression, this difference was not evidenced by traditional frequentist statistics, the phenotype did not clearly mimic ncc69 knockdown, nor was a difference seen in the mean peak magnitude of CT response. In totality, these results may suggest that CIII Cl- homeostasis involves other cotransporters which can adapt in either function or expression in response to loss or gain of function. Given the importance of this system to behavior selection, CIII Cl- homeostasis will make an interesting target for future experimentation (Himmel, 2023).
Importantly, this study has shown that overexpression of ncc69-a fly orthologue of NKCC1-is sufficient for driving a sensitization of cold nociception in larvae. As dysregulation of NKCC1 and kcc is associated with neuropathic pain in mammals, it is posited that altered larval cold nociception constitutes a new system in which to study neuropathic pain. Importantly, this system is wholly genetic and does not require injury or other methods of invoking nociceptive sensitization, making it a high throughput and easily accessible tool. Interestingly, RNAi knockdown of kcc did not mirror the ncc69 overexpression phenotype. It is speculated that this is because native kcc expression levels are low enough that knockdown does not sufficiently disrupt Cl- homeostasis. This might also be because of hypothetical unknown mechanisms of compensation, as discussed above (Himmel, 2023).
While it has been often stated that neuropathic pain is maladaptive, there is growing support for the hypothesis that neuropathic pain has its mechanistic bases in adaptive nociceptive sensitization-a mechanism by which organisms are more readily able to respond to danger following insult. Nerve injury has been previously shown to cause nociceptive sensitization in adult Drosophila and has been hypothesized to be protective. Moreover, it has been recently shown that hyperexcitability of CIII neurons is coincident with cold acclimation, the mechanism by which insects adapt to dips in temperature. One speculative hypothesis is that changes in expression levels of SLC12 transporters underlie these shifts in cold acclimation-induced cold sensitivity. This would be consistent with a study demonstrating that a number of genes in Drosophila involved in ion homeostasis are differentially regulated following cold acclimation. If this speculation is veridical, insect thermal acclimation may serve as an example of how ‘maladaptive’ injury and neuropathic sensitization can confer an adaptive advantage. It is therefore possible that these findings, and continued study, will lead to not only advances relevant to human health but also better understanding of nervous system evolution and the evolution of mechanisms underlying neuropathic sensitization and pain (Himmel, 2023).
In Drosophila, the clock that controls rest-activity rhythms synchronizes with light-dark cycles through either the blue-light sensitive cryptochrome (Cry) located in most clock neurons, or rhodopsin-expressing histaminergic photoreceptors. This study shows that, in the absence of Cry, each of the two histamine receptors Ort and HisCl1 contribute to entrain the clock whereas no entrainment occurs in the absence of the two receptors. In contrast to Ort, HisCl1 does not restore entrainment when expressed in the optic lobe interneurons. Indeed, HisCl1 is expressed in wild-type photoreceptors and entrainment is strongly impaired in flies with photoreceptors mutant for HisCl1. Rescuing HisCl1 expression in the Rh6-expressing photoreceptors restores entrainment but it does not in other photoreceptors, which send histaminergic inputs to Rh6-expressing photoreceptors. These results thus show that Rh6-expressing neurons contribute to circadian entrainment as both photoreceptors and interneurons, recalling the dual function of melanopsin-expressing ganglion cells in the mammalian retina (Alejevski, 2019).
The Drosophila sleep–wake rhythms are controlled by a brain circadian clock that includes about 150 clock neurons. Light synchronizes the clock neuronal network through cell-autonomous and non-cell-autonomous light input pathways. Cry is a blue-light sensitive photoreceptor protein that is expressed in most clock neurons. In the absence of Cry, flies do not phase-shift their behavioral rhythms in response to a short light pulse but still synchronize to light–dark (LD) cycles. Only flies devoid of both Cry and rhodopsin-expressing photoreceptors fail to entrain to LD cycles. Six different rhodopsins (Rhs) have been characterized in the Drosophila photoreceptive structures, which include the compound eye, the Hofbauer-Buchner (H-B) eyelet, and ocelli. The compound eye strongly contributes to circadian photoreception, whereas a modest contribution appears to be brought by the H-B eyelet and the ocelli. A circadian function has been recently associated with the yet poorly characterized rhodopsin 7, although its exact contribution and localization in the brain and/or the eye remains controversial. In addition to entrainment, the visual system controls other features of the clock neuron network by conveying light information to either promote or inhibit the behavioral output of specific clock neuron subsets (Alejevski, 2019).
The compound eye includes about 800-unit eyes (ommatidia), each of which contains eight photoreceptors. The six Rh1-expressing outer photoreceptors (R1–6) are involved in motion detection and project to the lamina neuropile of the optic lobe. The two inner photoreceptors (R7–8) are important for color detection and project to the medulla. They express four different rhodopsins and thus define two types of ommatidia: 'pale' (p) ommatidia (30%) include a Rh3-expressing R7 and a Rh5-expressing R8, whereas 'yellow' (y) ommatidia (70%) include a Rh4-expressing R7 and a Rh6-expressing R8. Each extra-retinal H-B eyelet contains four Rh6-expressing photoreceptors that project to the accessory medulla, in the vicinity of key pacemaker neurons, the ventral lateral neurons (LNvs) that produce the pigment-dispersing factor (PDF) neuropeptide9,20–24. Each of the three ocelli contains about 80 photoreceptors that express Rh225. The Drosophila rhodopsins cover a wide range of wavelengths from 300 nm to 600 nm18,19, with only Rh1 and Rh6 being sensitive to red light (Alejevski, 2019).
Rhodopsin-dependent circadian entrainment involves two downstream signaling pathways, the canonical one that relies on the phospholipase C encoded by the no receptor potential A gene (norpA)2 or an unknown pathway that does not contribute in very low light levels. All but Rh2- and Rh5- expressing photoreceptors support synchronization in very low light, and at least Rh1, Rh5, and Rh6 can signal through the NorpA-independent pathway. Photoreceptors of the compound eye are histaminergic but the H-B eyelet expresses both histamine and acetylcholine. Although the two neurotransmitters might contribute to circadian entrainment, flies devoid of Cry and histidine decarboxylase do not synchronize their rest–activity rhythms with LD cycles. This suggests that besides Cry, there is no histamine-independent pathway to entrain the clock (Alejevski, 2019).
Two genes encoding histamine-gated chloride channels, ora transientless (ort) and Histamine-gated chloride channel subunit 1 (HisCl1), have been identified in Drosophila. The ort-null mutants are visually blind and their electroretinograms have no ON and OFF transients. In contrast, HisCl1 mutants show increased OFF transients, whereas slower responses were observed in the postsynaptic laminar monopolar cells. Based on transcriptional reporters, ort expression in the optic lobes was observed in neurons of both the lamina and medulla/lobula neuropils. Based on reporter gene expression, HisCl1 was localized in glial cells of the lamina. However, recent work reported expression in photoreceptors, in particular in the R7 and R8 inner photoreceptor subtypes. Indeed, Ort and HisCl1 support color opponency between the two subtypes of 'inner' photoreceptors, the ultraviolet (UV)-sensitive R7 and non-UV-sensitive R8, with HisCl1 and Ort mediating direct and indirect inhibition, respectively. The histaminergic pathways that are involved in circadian entrainment are unknown and are the subject of the present study. The results show that both Ort and HisCl1 define two different pathways for circadian entrainment. Whereas Ort contributes through its expression in the interneurons of the optic lobe, HisCl1 mostly contributes through its expression in the Rh6-expressing retinal photoreceptors. The work thus reveals that Rh6-expressing neurons contribute to light-mediated entrainment as both photoreceptors and interneurons (Alejevski, 2019).
This work reveals that the Cryptochrome-independent entrainment of rest–activity rhythms relies on distinct pathways that are contributed by the two histamine receptors Ort and HisCl1. Whereas Ort mediates circadian entrainment through the optic lobe interneurons that are involved in visual functions, HisCl1 defines a new photoreceptive pathway through Rh6-expressing photoreceptors. Although both receptors mediate synchronization with a shifted LD cycle, it seems likely that the two pathways will show differences in specific light conditions. It was not possible to rescue Ort function with HisCl1 expression in the ort-expressing cells, whereas the Ort could replace HisCl1 in Rh6 photoreceptors. It is possible that HisCl1 has a lower affinity for histamine with Rh6 cells receiving more neurotransmitter than optic lobe interneurons. Alternatively, interneurons could sufficiently differ from photoreceptors for their physiology or specific receptor-interacting protein content, preventing HisCl1 from working efficiently. HisCl1 downregulation in Rh6 cells slows down synchronization and flies with HisCl1134 mutant eyes synchronize very poorly with advanced LD cycles, and fail to synchronize with delays. It cannot be excluded that non-photoreceptor cells contribute to HisCl1-dependent entrainment but other pathways appear to have a modest contribution if any (Alejevski, 2019).
HisCl1 is expressed in the H-B eyelet, which could thus contribute to this synchronization pathway. However, the cell-killing experiments indicate that H-B eyelet is not required for HisCl1-mediated synchronization through Rh6 cells. In the recently described color opponency mechanism, retinal R7 cells inhibit R8 and vice versa through HisCl1 expression in the photoreceptors (Schnaitmann, 2018). It is supposed that HisCl1-dependent clock synchronization is also mediated by the hyperpolarization of Rh6-expressing cells. How this hyperpolarization interacts with the light-induced depolarization in Rh6 photoreceptors to result in a synchronization message to the clock neurons remains to be understood. Since only Rh6-expressing R8 and not the other inner photoreceptors contribute to this circadian photoreception pathway, Rh6 cells might have specific connections with downstream interneurons. Such specificity has been described for color vision where each of the four inner photoreceptor subtypes connects to a different type of TmY interneuron in the Medulla. This study shows that HisCl1 expression in Rh6 cells supports synchronization with red light, in the absence of Rh1, indicating that an intra-Rh6-photoreceptor circuit is sufficient. This indicates that Rh6-expressing R8 photoreceptors play a dual photoreceptor/interneuron role in this pathway (Model for the retinal input pathways to the brain clock). Whether the same individual cells have the two roles is unknown, although the HisCl1-dependent color opponency mechanism suggests that it could be the case. It is also unclear whether all Rh6-expressing R8 photoreceptors or only a fraction of them contribute to circadian synchronization. The results imply that, in addition to histaminergic neurotransmission, Rh6-expressing photoreceptors can talk to downstream interneurons through histamine-independent neurotransmission. A recent transcriptomics study indeed revealed the expression of cholinergic markers in R7 and R8 cells, supporting cholinergic transmission in the inner photoreceptors, in addition to histaminergic transmission (Alejevski, 2019).
The data indicate that histaminergic inputs from both outer and inner photoreceptors converge to Rh6 cells to contribute to circadian entrainment. It is possible that some of these inputs rely on Rh7, which seems to be expressed in Rh6-expressing photoreceptors, according to transcriptional reporter data. Putative connections between photoreceptors have been described in Drosophila and other insects. How R1–6 photoreceptors might be connected to Rh6-expressing R8 cells remains difficult to understand, but a few putative contacts between presynaptic outer cells and postsynaptic inner cells have been observed in Musca. The intra-retinal functional connectivity that this study reports in Drosophila is reminiscent to the circuit logic of circadian entrainment in the mammalian retina, where intrinsically photoreceptive retinal ganglion cells express the melanopsin photopigment in addition to receiving inputs from rods and cones. Interestingly, melanopsin appears to share light-sensing properties with the rhabdomeric photoreceptors of invertebrates. It has been shown that the mammalian circadian clock can synchronize with day–night cycles by tracking light color changes in addition to light intensity changes. It will be interesting to investigate the possible contribution of the dual function of Rh6-expressing photoreceptors to integrating different color cues into the retinal information that is sent to the clock (Alejevski, 2019).
Mutations in MPV17 are a major contributor to mitochondrial DNA (mtDNA) depletion syndromes, a group of inherited genetic conditions due to mtDNA instability. To investigate the role of MPV17 in mtDNA maintenance, this study generated and characterized a Drosophila melanogaster Mpv17 (dMpv17) KO model showing that the absence of dMpv17 caused profound mtDNA depletion in the fat body but not in other tissues, increased glycolytic flux and reduced lifespan in starvation. Accordingly, the expression of key genes of glycogenolysis and glycolysis was upregulated in dMpv17 KO flies. In addition, it was demonstrated that dMpv17 formed a channel in planar lipid bilayers at physiological ionic conditions, and its electrophysiological hallmarks were affected by pathological mutations. Importantly, the reconstituted channel translocated uridine but not orotate across the membrane. These results indicate that dMpv17 forms a channel involved in translocation of key metabolites and highlight the importance of dMpv17 in energy homeostasis and mitochondrial function (Corra, 2023).
Receptors for bitter, sugar, and other tastes have been identified in the fruit fly Drosophila melanogaster, while a broadly tuned receptor for the taste of acid has been elusive. Previous work showed that such a receptor was unlikely to be encoded by a gene within one of the two major families of taste receptors in Drosophila, the ‘gustatory receptors' and ‘ionotropic receptors.' To identify the acid taste receptor, this study tested the contributions of genes encoding proteins distantly related to the mammalian Otopertrin1 (OTOP1) proton channel that functions as a sour receptor in mice. RNA interference (RNAi) knockdown or mutation by CRISPR/Cas9 of one of the genes, Otopetrin-Like A (OtopLA), but not of the others (OtopLB or OtopLC) severely impaired the behavioral rejection to a sweet solution laced with high levels of HCl or carboxylic acids and greatly reduced acid-induced action potentials measured from taste hairs. An isoform of OtopLA that was isolated from the proboscis was sufficient to restore behavioral sensitivity and acid-induced action potential firing in OtopLA mutant flies. At lower concentrations, HCl was attractive to the flies, and this attraction was abolished in the OtopLA mutant. Cell type-specific rescue experiments showed that OtopLA functions in distinct subsets of gustatory receptor neurons for repulsion and attraction to high and low levels of protons, respectively. This work highlights a functional conservation of a sensory receptor in flies and mammals and shows that the same receptor can function in both appetitive and repulsive behaviors (Ganguly, 2021).
The functional conservation of the Otop channels for acid taste in flies is striking given that chemosensory receptors tend to vary greatly in flies and mammals, which diverged ∼800 million y ago. In contrast to the Otop channels, the two major families of fly receptors (GRs and IRs), which function in tasting sugars, bitter compounds, acetic acid, amino acids, polyamines, N, N-diethyl-meta-toluamide (DEET), CO2, and other tastants are not present in mammals. The retention of Otop channels for acid taste in flies and mice is remarkable since the gross anatomies of the gustatory systems are very different. In addition, the taste receptor cells in flies are neurons, while they are modified epithelial cells in mammals (Ganguly, 2021).
The conserved role for Otop proteins for acid taste in flies and mammals cannot be explained by greater selective pressure for maintaining a receptor for a mineral (e.g., H+) versus organic molecules since other minerals (Ca2+ and Na+) are sensed in flies through IRs, which are not present in mammals. Thus, the retention of Otop channels for acid taste in flies and mammals underscores the very strong selection for this acid sensor for animal survival. Otop-related proteins are encoded in many distantly related terrestrial and aquatic vertebrates ranging from the platypus to frogs and pufferfish, as well as ancient invertebrates such as worms and insect disease vectors, including Aedes aegypti. Thus, despite the considerable diversity of most chemosensory receptors, it is plausible that Otop channels endow a large proportion of the animal kingdom with acid taste (Ganguly, 2021).
A question concerns the cellular mechanism through which the sensation of protons is detected. OtopLA is expressed in the four classes of GRNs in taste hairs (A to D). The B and D GRNs respond to aversive tastants (B, bitter, high Na+ etc; D, Ca2+, high Na+, K+), while the A and C GRNs are activated by chemicals that stimulate consumption (A, sugars, low Na+ fatty acids, etc; C, water). The data indicate that both B and D GRNs contribute to acid repulsion but that B GRNs comprise the major class required for acid repulsion, while D GRNs are the minor class. In support of this conclusion, RNAi knockdown of B but not D GRNs impaired acid repulsion. In addition, we fully rescued the OtopLA1 mutant phenotype by expression of the OtopLAp transgene in B GRNs but only partially rescued the deficit by expression of OtopLAp in D GRNs. In addition, our data indicate that both A and C GRNs contribute to the modest attraction to 0.01 HCl in wild-type flies. This attraction is eliminated in the OtopLA1 mutant. The C GRNs may be more important, as expression of the OtopLAp transgene in A GRNs reduced the impairment in the mutant, but the suppression of the phenotype fell below the threshold for statistical significance (Ganguly, 2021).
It has been reported that acids cause repulsion of sugary foods by direct activation of B GRNs and suppression of sugar-induced activation of A GRNs. This previous study focused on behavioral responses to carboxylic acids, and this study repeated this finding for citric acid. However, at the cellular level, when the pH of sucrose was decreased, no reduced sucrose-induced action potentials were induced. Thus, it is concluded that protons do not suppress A GRNs. Rather, it is suggest that A GRNs are inhibited by certain organic anion moieties of carboxylic acids. A mechanism by which the activities of both A GRNs and B GRNs are affected by carboxylic acids, but only B GRNs by protons could provide a coding mechanism for differentiating between protons and carboxylic acids (Ganguly, 2021).
Following submission of the initial version of this manuscript, another group also reported a role for OtopLA in acid taste in Drosophila (Mi, 2021). These researchers found that OtopLA is required for attractive responses to low concentrations of acids, as did this study, but not for aversive responses to higher concentrations of acids. However, even in wild-type controls, they did not observe significant repulsion until the pH was reduced to high levels (≤2) that may be damaging to cells. At these very low pHs, the nociceptive response is likely to have a major contribution to avoidance. Mi (2021). also reported that the flies exhibited a much higher level of attraction to acids than what was observed in the current study. The differences in levels of attraction and repulsion might be due to variations in fly food between laboratories, the precise ages of the flies, hours of starvation, or a combination of these factors. Nevertheless, although the level of acid attraction differs, both studies find that the deficit in attraction in OtopLA mutants can be suppressed by expression of a wild-type transgene in A GRNs. In addition, this study found that this phenotype is suppressed by expression of the OtopLA rescue transgene in C GRN (Ganguly, 2021).
Another difference between the current report and that of Mi (2021) is that they reported that expression of OtopLAa in human embryonic kidney 293 (HEK293) cells led to the appearance of inward currents in response to acid stimuli (pH range 6 to 3). It has been previously demonstrated that both vertebrate and invertebrate Otop proteins form proton channels. However, this study did not observe any acid-induced currents using stimuli as low as pH 3.0 for either OtopLAp or OtopLAa (FBgn0259994) expressed in either HEK293 cells or Xenopus oocytes, even though surface expression was detected when the channels were tagged with GFP. There are several possible reasons why the Drosophila OtopLA channel did not generate functional currents in either cell type. One possibility is that the native system provides factors or binding partners necessary to gate the channels. It is noted that OtopLA is the only one of the Drosophila Otop channels to have a large extracellular domain between transmembrane domains 5 and 6, which might bind ligands or proteins (Ganguly, 2021).
Together, these data point to a complex role of the OtopLA channel in the gustatory system of Drosophila, where it is expressed in multiple types of sensory cells and can mediate both attractive and aversive responses. Interestingly, humans also find acids appetitive at low concentrations and aversive at higher concentrations. The elucidation of the cellular and molecular mechanism of acid-sensing that we describe here can serve as the basis for further understanding as to how animals assign valence to stimuli that vary only in intensity (Ganguly, 2021).
Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dβ1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dβ1 nAChRs when expressed in X. laevis oocytes. This study investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dβ1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dβ1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dβ1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dβ1 (Dα1/Dβ1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1/Dβ1 orthosteric site over the Dα1/Dβ1 orthosteric site (Takayama, 2022).
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