InteractiveFly: GeneBrief

inebriated: Biological Overview | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

The inebriated (ine) mutation was originally identified on the basis of increased neuronal excitability (Stern, 1992). Double mutants defective in both ine and Shaker (Sh), which encodes a potassium channel alpha subunit channel, display a 'downturned wings and indented thorax phenotype'. This appearance is identical to Sh mutants carrying either an additional mutation in eag, which encodes a potassium channel alpha subunit, or carrying a duplication of the para gene (termed Dp para⁺), which encodes a sodium channel (X. Huang, 2002 and Y. Huang, 2002 and references therein).

Mutants defective in ine exhibit a second phenotype resulting from increased neuronal excitability -- an increased rate of onset of the so-called 'long-term facilitation' at the larval neuromuscular junction (NMJ). Long term facilitation is an adaptation of the neuron in which continued nerve stimulations elicit motor nerve depolarizations that are increased in duration. This ine phenotype is also exhibited by several additional mutants in which neuronal excitability is increased. These phenotypes suggest that ine mutations increase neuronal excitability by reducing K currents or increasing Na currents. The ine gene encodes two protein isoforms, Ine-P1 and Ine-P2, which share high homology to members of the Na⁺/Cl^--dependent neurotransmitter transporter family. Ine-P1 is identical to Ine-P2, except that it contains 300 additional amino acids in the N-terminal intracellular domain. ine mutations appear to cause increased excitability of the Drosophila motor neuron by causing the defective reuptake of the substrate neurotransmitter of the ine transporter and thus overstimulation of the motor neuron by this neurotransmitter (X. Huang, 2002 and Y. Huang, 2002 and references therein).

Ine functions in the short-term (time scale of minutes to a few hours) to regulate neuronal excitability. Ine is able to control excitability from either neurons or glia cells. Overexpression of Ine reduces neuronal excitability. Overexpression phenotypes of ine include delayed onset of long-term facilitation and increased failure rate of transmitter release at the larval neuromuscular junction; reduced amplitude of larval nerve compound action potentials; suppression of the leg-shaking behavior of mutants defective in the Shaker-encoded potassium channel, and temperature-sensitive paralysis. Each of these overexpression phenotypes closely resembles those of loss of function mutants in the para-encoded sodium channel. These data raise the possibility that Ine negatively regulates neuronal sodium channels, and thus that the substrate neurotransmitter of Ine positively regulates sodium channels (Y. Huang, 2002).

Ine also appears to regulate the osmotic stress response by regulating osmolyte transport within the Malpighian tubule and hindgut (Chiu, 2000; X. Huang, 2002). Water reabsorption by organs such as the mammalian kidney and insect Malpighian tubule/hindgut requires a region of hypertonicity within the organ. To balance the high extracellular osmolarity, cells within these regions accumulate small organic molecules called osmolytes. These osmolytes can accumulate to a high level without toxic effects on cellular processes. ine mutants lacking both isoforms are hypersensitive to osmotic stress, which was assayed by maintaining flies on media containing NaCl, KCl, or sorbitol: this hypersensitivity is completely rescued by high-level ectopic expression of the ine-RB isoform. Evidence is provided that this hypersensitivity represents a role for ine that is distinct from the increased neuronal excitability phenotype of ine mutants. Both wild-type and ine mutants exhibit a 'threshold' response to osmotic stress: for each genotype, maintenance on media containing an [NaCl] above this threshold causes inviability. Relative contributions to the osmotic stress response of each of the two isoforms of ine has been assessed. Each protein isoform, when independently overexpressed with the GAL4 system, can rescue the osmotic stress response defect of ine mutants, suggesting that the two isoforms have similar functions. However, Ine-P2 alone, when expressed from its normal chromosomal position, is sufficient for only a small degree of resistance to osmotic stress. These results demonstrate a role for the ine-encoded transporters in the osmotic stress response (X. Huang, 2002).

When cells are placed in a hypertonic media, an extremely rapid loss of water is followed by influx of Na+ and K+. This influx of ions causes the passive return of water to the cell, thus enabling cell volume to be recovered. However, this influx also increases the intracellular [Na+] and [K+] with detrimental consequences to the activity of essential cellular functions. To accommodate to osmotic stress, cells then replace the intracellular Na+ and K+ with nonperturbing osmolytes such as betaine, sorbitol, inositol, or glycerol. This replacement thus enables a restoration of normal cell volume and normal intracellular [Na+] and [K+]. It is proposed that the Ine transporter plays an important role in this replacement by enabling the transport of an osmolyte, the substrate for the Ine transporter, which has not yet been identified. Thus, in ine mutants this replacement fails to occur properly, and, following osmotic stress, the elevated Na+ and K+ levels within hindgut epithelial cells persist for the duration of the exposure to osmotic stress. Alternatively, Ine, functioning in glial cells may simply regulate K+ absorption (Chiu, 2000) and it might not function as a transporter of an unknown osmolyte. Chiu cloned MasIne, the inebriated homolog from Manduca sexta, and reported two isoforms: a short form and a long form containing an additional 108 amino acids at the N terminus. Injection of the long form, but not the short form, into Xenopus oocytes elicited a hyperosmolarity-induced Cl- current, which was attributed to a phospholipase C-mediated activation of a Ca2+ flux. Furthermore, the additional 108 amino acids in MasIne-long is sufficient to confer a similar Cl- current when appended to the GABA transporter GAT1. Thus, Chiu suggests a role for Ine in response to hyperosmolarity, although the mechanism that they suggest (induction of a Ca2+-activated K+ channel) is quite different from the mechanism of osmolyte accumulation proposed by Y. Huang {2002). One possibility is that Ine and MasIne utilize different mechanisms to respond to hyperosmolarity. The observation that the 108-amino-acid domain of MasIne-long shares only 9 amino acids with Ine-P1 is consistent with this possibility. Alternatively, Ine and MasIne might each use both mechanisms to respond to hyperosmolarity. Further research will be required to distinguish between these possibilities (X. Huang, 2002).

DEVELOPMENTAL BIOLOGY

In situ hybridization of ine transcripts to developing embryos has revealed expression of this gene in several cell types, including the posterior hindgut, Malpighian tubules, anal plate, garland cells, and a subset of cells in the central nervous system. During germ-band extension (stage 9), the primordium of the hindgut shows elevated levels of transcripts. During germ-band retraction (stage 13), the midgut, Malpighian tubules, garland cells, anal plate, and foregut also express transcripts, and specific hybridization to head regions becomes apparent. Central nervous system staining is segmentally repeating in cells flanking the midline of the ventral ganglion. This central nervous system expression pattern is similar to the dSERT expression pattern. In contrast to ine, however, there was no nonneuronal expression reported for dSERT (Soehnge, 1996).

Tissue in situ experiments determined that the ine transcript is localized to many tissues, with higher levels of hybridization in the nervous system and digestive tract (Burg, 1996).

The extremely long N-terminal intracellular domain observed in Ine-P1 is not commonly observed in members of this transporter family. This observation raises the possibility that this extended intracellular domain reflects an additional Ine activity distinct from neurotransmitter transport. If so, then Ine-P1 and Ine-P2 might perform distinct functions in Drosophila and thus might exhibit different expression patterns. To test this possibility, in situ hybridization probes were constructed that were specific for either the ine-RA or the ine-RB cDNAs. The embryonic expression patterns of the two cDNAs are virtually indistinguishable, suggesting that the two cDNAs function in the same cells (X. Huang, 2002).

Functional analysis of Ine

The inebriated homolog MasIne has been cloned from Manduca sexta and has been expressed in Xenopus laevis oocytes. MasIne is homologous to neurotransmitter transporters but no transport was observed with a number of putative substrates. Oocytes expressing MasIne respond to hyperosmotic stimulation by releasing intracellular Ca(2+), as revealed by activation of the endogenous Ca(2+)-activated Cl(-) current. This Ca(2+) release requires the N-terminal 108 amino acid residues of MasIne and occurs via the inositol trisphosphate pathway. Fusion of the N terminus to the rat gamma-aminobutyric acid transporter (rGAT1) also renders rGAT1 responsive to hyperosmotic stimulation. Immunohistochemical analyses show that MasIne and Drosophila Ine have similar tissue distribution patterns, suggesting functional identity. Inebriated is expressed in tissues and cells actively involved in K(+) transport, which suggests that it may have a role in ion transport, particularly of K(+). It is proposed that stimulation of MasIne releases intracellular Ca(2+) in native tissues, activating Ca(2+)-dependent K(+) channels, and leading to K(+) transport (Chiu, 2000).

MasIne and Drosophila Ine are highly homologous to the neurotransmitter transporter family of proteins. However, phylogenetic analysis shows that the Inebriated proteins are divergent from other neurotransmitter transporters, suggesting that they have a common, yet distinct, function from that of the other transporters. MasIne could be expressed in the plasma membrane of Xenopus oocytes, but none of the ligands and/or substrates tested was transported. A lack of transport could also result either because the correct substrate could not be identified or because an additional protein may be required for transport. However, these data show that inebriated is not a GABA or glutamate transporter, as proposed by Soehnge (1996). Indeed, these transporters have been cloned and are distinct proteins (Chiu, 2000 and references therein).

Nevertheless, oocytes expressing MasIne displayed ligand-independent leakage conductances to alkali ions, suggesting that MasIne shares this property with other transporters. However, Na⁺-coupled, ligand-independent transient currents, which are often seen with other neurotransmitter transporters, were not observed (Chiu, 2000).

Although ligand transport was not observed in Xenopus oocytes with the substrates analyzed, MasIne responds to hyperosmotic stimulation by modulating the activation of outward Cl^- currents. Using pharmacological agents, these Cl^- currents were shown to be dependent on the release of intracellular Ca²⁺ through a PLC and InsP3 signaling pathway. Moreover, the current observed, I_Cl(Ca), display a voltage- and time-dependence similar to those induced by InsP3 injection of oocytes. This hyperosmotic-sensitive Cl^- current was observed in Xenopus oocytes when the full-length MasIne protein, MasIne-135, was expressed. No Cl^- currents were observed with expression of MasIne-459 or with the GABA and serotonin transporters during hyperosmotic stimulation (Chiu, 2000).

Expression of a fusion protein consisting of the MasIne-135 N terminus with rGAT1 resulted in spontaneous activation of I_Cl(Ca), even without hyperosmotic stimulation. These currents were also observed when the N terminus alone was expressed in oocytes without osmotic stimulation. These data suggest that the N-terminal 108 amino acid residues of MasIne modulate PLC activation (Chiu, 2000).

Since the N terminus spontaneously activates endogenous currents, the data indicate that, when the full-length protein is expressed, the N terminus interacts with other MasIne domains and, therefore, is unable to activate these currents. Hyperosmotic stimulation releases this N terminus, leading to PLC activation. In contrast, both 108-rGAT1 and DMasIne-135 lack this 'negative regulator' so that spontaneous activation of PLC occurs. Moreover, water-injected oocytes can also respond to hyperosmotic stimulation, but this response is significantly delayed (by more than 1 min) and it occurs at much higher osmolarities. This observation indicates that oocytes contain an endogenous hyperosmotic-responsive system that activates I_Cl(Ca) (Chiu, 2000).

Although none of the compounds analyzed were transported, it is speculated that MasIne is a transporter. The transport of an unknown ligand, which could be an osmolyte, may cause activation of the PLC/InsP3 cascade. This activation, dependent on the MasIne N terminus, is apparently not mediated through a G-protein. Furthermore, since this N terminus has no homology with either Ga or protein tyrosine kinases, both known PLC activators, it appears that PLC activation is mediated through a novel mechanism. Activation of the PLC/InsP3 cascade leads to increases in intracellular Ca²⁺ concentrations, which can then stimulate Ca²⁺-activated channels, including those that transport K⁺ (Chiu, 2000).

The Inebriated protein is expressed in nearly identical tissue patterns in the nervous and muscular systems of both Manduca sexta and Drosophila melanogaster. However, differences in the expression patterns are observed in the gut, perhaps reflecting dietary differences between the two insect species. Manduca sexta larvae feed on a diet high in K⁺, which is used for nutrient transport in the midgut. To maintain homeostasis, K⁺ in the hemolymph is transported into the midgut by a coupled K⁺/2H⁺ transport system. In the midgut, the V-ATPase in goblet cell apical membranes pumps H⁺ from the cytoplasm into the goblet cell cavity. The protons are exchanged with K⁺ by a K⁺/2H⁺ exchanger, resulting in net K⁺ transport from the goblet cell into the midgut. Both passive and active K⁺ transport processes are present in the goblet cell basolateral membrane, indicating that several mechanisms of K⁺ transport exist in this membrane. It is speculated that MasIne, localized entirely in the basolateral membrane of goblet cells, may play a role in K⁺ transport from the hemolymph into the goblet cells (Chiu, 2000).

In lepidopteran insects, such as Manduca sexta, the Malpighian tubules, ileum, rectum and cryptonephridial system, a sac-like structure that packs Malpighian tubules tightly with the rectal epithelium, are all involved in the maintenance of salt and water balance in the hemolymph. In these tissues, MasIne is expressed at high levels only in regions involved in ion reabsorption. The middle region of Malpighian tubules is not specialized for ion reabsorption, and MasIne expression in this region is very low. The glial cell layer and its processes form a blood-brain barrier around the neurons and neuropil, which constantly secretes Na+ and absorbs K+, so that high hemolymph K+ levels do not affect neuronal function. Potentially, MasIne in glial cells may regulate K+ absorption. MasIne expression in the axonal plasma membrane suggests that it could also modulate neuronal excitability by stimulating Ca²⁺-sensitive channels that cause membrane repolarization (Stern, 1992). This is the first report demonstrating the involvement of the PLC/InsP3 signaling cascade in a member of the Na⁺/Cl^--dependent neurotransmitter transporter family. The mechanisms by which these processes are modulated are not known, and additional studies are needed to define the mechanisms involved more fully (Chiu, 2000).

Because the electrophysiological defects of ine mutants are observed in motor neurons (Stern, 1992), targeted ine expression only in neurons could be sufficient for rescue. Alternatively, ine could exert its effects on neuronal excitability from glial cells; often, transporters that perform reuptake of neurotransmitter released from neurons are located in neighboring glia. Finally, perhaps expression in either cell type could be sufficient for rescue. The latter possibility would be consistent for a neurotransmitter transporter, which acts on neurotransmitters in the extracellular space between adjacent cells. To test these possibilities, ine-RA expression was targeted either to neurons or to specific glia with specific GAL4 drivers and the UAS-ine-RA line (Y. Huang, 2002).

Two GAL4 lines were used to target Ine-P1 expression to different subsets of glial cells. The MZ1580 line expresses Gal4 from stage 11 in the longitudinal glioblast and its progeny, and later in most other glial cells. The gli-gal4 line expresses the Gal4 protein specifically in peripheral glial cells, which wrap the motor and sensory axons of peripheral nerves. Expression of Ine-P1 from an UAS-ine-RA construct driven by either of these GAL4 lines is able to rescue fully both the downturned wings phenotype and the increased rate of onset of long-term facilitation phenotype. The rescued lines require even more repetitive nerve stimulation than wild type for the onset of long-term facilitation. This observation raised the possibility that overexpression of ine with the GAL4 system could reduce neuronal excitability. These results indicate that Ine-P1 can function effectively from glial cells (Y. Huang, 2002).

Glial cells seem to be a more favorable site for Ine function, because targeted expression of ine in the peripheral glia fully rescues the ine mutant phenotypes, whereas targeted expression of ine in neurons using the elav-gal4 driver only partially rescues the mutant phenotypes (Y. Huang, 2002).

ine expresses two transporter isoforms, Ine-P1 and Ine-P2. The N terminal intracellular domain of Ine-P1 is ~300 amino acids longer than that of Ine-P2; the two isoforms are otherwise identical. Transcripts of the two isoforms are colocalized in both the nervous system and the fluid reabsorption system of the flies (Soehnge, 1996; X. Huang, 2002). An N-terminal domain of the length of Ine-P1 is unusual for a member of this protein family and raises the possibility that this domain performs a function unrelated to neurotransmitter transport that is required for the control of neuronal excitability. If so, then Ine-P2, which lacks this extended N terminus, might be unable to function in the absence of Ine-P1. To test this possibility, Ine-P2 was expressed in glia by using the MZ1580 GAL4 line to drive expression of UAS-ine-RB. Unlike Ine-P1, which fully rescues the ine phenotypes, Ine-P2 rescues the ine phenotypes only partially. For example, 94% of the Sh;ine flies carrying MZ1580 and UAS-ine-RA were rescued for the downturned wings phenotype, whereas only 39% of the Sh;ine flies carrying MZ1580 and UAS-ine-RB exhibit rescue. Similarly, ine mutant larvae carrying both MZ1580 and UAS-ine-RB exhibit only a partial rescue of the increased rate of onset of long-term facilitation: this degree of rescue was significantly different from the extent of rescue of ine mutants carrying both MZ1580 and UAS-ine-RA. Thus, the presence of Ine-P2 alone provides some ine activity, but Ine-P2 alone is much less effective than Ine-P1 alone (Y. Huang, 2002).

This long N terminus of Ine-P1 is uncommon among members of the Na⁺/Cl^--dependent neurotransmitter transporter family and raises the possibility that this domain might perform a function that is distinct from neurotransmitter transport but is required for the control of neuronal excitability. If so, then Ine-P2, which lacks the long N terminal intracellular domain, would be unable to perform this function and would be unable to confer any ine⁺ activity in the absence of Ine-P1. The demonstration that each isoform is able to perform ine⁺ function on its own does not support this possibility. The Ine-P2 isoform performs less effectively than Ine-P1, which raises the possibility that the long N terminus of the Ine-P1 isoform might be required for efficient transporter activity. For example, the N terminus might be required for proper localization, stability, or activation of the transporter (Y. Huang, 2002).

The putative Na(+)/Cl(-)-dependent neurotransmitter/osmolyte transporter Inebriated in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis

Most organisms are able to maintain systemic water homeostasis over a wide range of external or dietary osmolarities. The excretory system, composed of the kidneys in mammals and the Malpighian tubules and hindgut in insects, can increase water conservation and absorption to maintain systemic water homeostasis, which enables organisms to tolerate external hypertonicity or desiccation. However, the mechanisms underlying the maintenance of systemic water homeostasis by the excretory system have not been fully characterized. The present study found that the putative Na(+)/Cl(-)-dependent neurotransmitter/osmolyte transporter inebriated (ine) is expressed in the basolateral membrane of anterior hindgut epithelial cells. This was confirmed by comparison with a known basolateral localized protein, the alpha subunit of Na(+)-K(+) ATPase (ATPalpha). Under external hypertonicity, loss of ine in the hindgut epithelium results in severe dehydration without damage to the hindgut epithelial cells, implicating a physiological failure of water conservation/absorption. It was also found that hindgut expression of ine is required for water conservation under desiccating conditions. Importantly, specific expression of ine in the hindgut epithelium can completely restore disrupted systemic water homeostasis in ine mutants under both conditions. Therefore, ine in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis (Luan 2015).

Water homeostasis is essential for the survival of all organisms. The mammalian kidney and the Malpighian tubule and hindgut of insects play indispensable roles in maintaining water homeostasis over a wide range of external or dietary osmolarities. These organs can increase water conservation and absorption to maintain systemic water homeostasis, which enables organisms to tolerate external hypertonicity or desiccation (Luan 2015).

The mammalian kidney regulates water balance mainly through the antidiuretic hormone (ADH), which enhances water absorption. Failure of antidiuretic mechanisms can result in disrupted systemic water homeostasis, causing pathological conditions like Diabetes Insipidus. Although antidiuretic factors for the enhancement of water absorption, such as Schgr-ITP and CAPA-related peptides, are also present in insects, the mechanisms of water conservation and absorption in the excretory system are not fully characterized, especially in Drosophila (Luan 2015).

Previous studies have shown that loss of the putative Na+/Cl--dependent neurotransmitter/osmolyte transporter ine causes hypersensitivity to dietary hypertonicity in Drosophila; however, the mechanism underlying this effect remains unknown. Ine is a member of the Na+/Cl--dependent neurotransmitter/osmolyte transporter family, which is conserved across invertebrates and vertebrates. Members of this family share several common structural features, including 12 transmembrane domains flanked by intracellular N and C termini, and an extracellular loop between the third and fourth transmembrane domains. These proteins play critical roles in neurotransmission, as well as cellular and systemic homeostasis, by transporting neurotransmitters, osmolytes, and energy metabolites across the plasma membrane. There is sequence similarity between ine and the betaine/GABA transporter (BGT1), a mammalian member of the Na+/Cl--dependent neurotransmitter/osmolyte transporter family. Both BGT1 and ine are expressed in the central nervous system (CNS), as well as organs that perform water absorption, and both are involved in the control of neuronal excitability and tolerance to hypertonicity. This suggests that these two proteins may function through a similar mechanism (Luan 2015).

Betaine, an active organic compound, is the substrate of BGT1 in renal medullary cells; however, the substrate of ine has yet to be identified. Betaine, like other intracellular organic osmolytes, can protect cells from external hypertonicity by balancing high extracellular osmolarity and preserving cell volume without interfering with cell function. However, no direct genetic evidence supports the osmoprotective function of the BGT1-mediated accumulation of betaine in renal medullary cells. Specifically, BGT1 knockout mice are healthy, and renal medullary cells appear to be normal in the hypertonic environment of the renal medulla. Therefore, the physiological function of the Na+/Cl--dependent neurotransmitter/osmolyte transporter in the excretory system remains to be elucidated (Luan 2015).

By investigating the function of ine in Drosophila, an excellent genetic model in which gene expression can be evaluated and manipulated in vivo, the physiological function will be better understood of Na+/Cl--dependent neurotransmitter/osmolyte transporters, including BGT1, in the excretory system. This study elucidates the role of ine in the Drosophila hindgut, and reveal a novel mechanism mediated by ine for the maintenance of systemic water homeostasis (Luan 2015).

This study has demonstrated that the mediation of water conservation/absorption by ine in the hindgut is essential for the maintenance of systemic water homeostasis in Drosophila. In insects, systemic water homeostasis is tightly regulated by the excretory system, including the Malpighian tubules and the hindgut, to ensure a constant internal environment. The dynamic balance between Malpighian tubule secretion and hindgut reabsorption, both of which are controlled by diuretic and antidiuretic hormones or factors, maintains water homeostasis in response to fluctuations in external osmotic conditions. However, in adult Drosophila, the water conservation/absorption mechanisms of the hindgut have not been elucidated. The current results demonstrate that ine is expressed in the basolateral membrane of the hindgut epithelium, suggesting that ine transports substrate from the hemolymph into hindgut epithelial cells. Surprisingly, under conditions of external hypertonicity, the systemic water homeostasis of ine mutant flies is disrupted, whereas that of WT flies is not disturbed. These results demonstrate that hindgut expression of ine mediates water conservation/absorption under external hypertonicity and maintains systemic water homeostasis. These results also suggest possible mechanism for ine function: transport of an osmolyte by ine into the hindgut epithelium increases intracellular molarity, which enhances water conservation/absorption from the hindgut lumen. Such a function would be particularly important in the condition of external hypertonicity, when increased molality in the hindgut lumen prevents osmotic flow of water into hindgut epithelium (Luan 2015).

It could be argued that ine functions through an osmoprotective mechanism, in which increased intracellular accumulation of osmolytes mediated by ine protects the hindgut epithelium from cellular death due to extracellular hypertonicity. However, this study demonstrates that anterior hindgut epithelial cells are not damaged by external hypertonicity in the absence of ine, suggesting that ine function in water conservation/absorption is not secondary to an osmoprotective effect. The existence of other osmolytes or transporters is proposed that function as osmoprotectors, and protect anterior hindgut epithelial cells against lethality under external hypertonicity. The expression of several genes, including some organic transporters, is up-regulated in the hindgut in response to external hypertonicity, supporting this possibility (Luan 2015).

Ine protein is expressed solely in the anterior hindgut. The anterior hindgut is an important site of water absorption, as demonstrated in insects other than Drosophila. In locusts, isosmotic fluid absorption in the anterior hindgut is driven by an apical membrane electrogenic Cl- pump. The antidiuretic hormone Schgr-ITP acts on the locust hindgut via cyclic AMP and GMP to increase the conductance of both K+ and Na+ and to stimulate the Cl- pump. As a result of the increased ion uptake, water absorption increases. It remains unknown, however, whether similar ion-uptake-coupled water absorption mechanisms are present in the Drosophila hindgut. This study found that loss of ine in the anterior hindgut epithelium causes severe dehydration in response to a hypertonic diet, and higher rates of body water loss under desiccation, which suggests the existence of a new mechanism of water conservation/absorption in the hindgut of Drosophila mediated by ine. It was proposed that ine transports osmolytes across the plasma membrane from the hemolymph and accumulates osmolytes within the hindgut epithelium, generating an osmotic driving force to conserve/absorb water from hindgut lumen against external hypertonicity. However, this theory lacks an explanation for how water is transferred into the hemolymph from epithelial cells, and to date, the transporter activity of ine has not been confirmed. The possibility thatine may improve water conservation/absorption through a different, unknown mechanism cannot be ruled out (Luan, 2015)

In addition to the anterior hindgut, the Malpighian tubules, rectum, and midgut also contribute to water absorption and conservation in insects under conditions of external hypertonicity or desiccation. During dehydration stress, the modulation of tyramine signaling in Drosophila Malpighian tubules enhances conservation of body water. Several anti-diuretic factors acting on the Malpighian tubules have been found. For example, CAPA-1 acts on Ncc69, the Na+-K+-2Cl- cotransporter, to increase water absorption through an ion uptake coupled mechanism. In addition, PKG, a cGMP-dependent kinase antagonizes the diuretic effects of tyramine and leukokinin. The rectum can also transport water from lumen to the hemolymph. In the locust, the chloride transport stimulating hormone (CTSH) acts to increase ion-dependent active transport of fluid from the rectum lumen. Finally, the antidiuretic hormone RhoprCAPA-2 inhibits fluid transport into the midgut lumen in Rhodnius prolixus to conserve water. Therefore, ine-mediated water conservation/absorption may not be the only mechanism by which systemic water homeostasis is maintained under external hypertonicity in Drosophila (Luan 2015).

Water is essential for the proper function of virtually all living cells. Organisms have developed mechanisms in the excretory system to maintain water hemostasis for a constant internal milieu under different external osmotic conditions, such as hypertonicity. This study reveals that hindgut expression of ine, a putative Na+/Cl-dependent neurotransmitter/osmolyte transporter, is indispensable for the maintenance of systemic water homeostasis in Drosophila. However, further investigation of the novel mechanism mediated by ine in the hindgut is necessary to fully understand the water conservation and absorption mechanisms of Drosophila hindgut, as well as the physiological functions of the members of the Na+/Cl-dependent neurotransmitter/osmolyte transporter family (Luan 2015)

EFFECTS OF MUTATION

On the basis of behavioral interactions with mutations in a potassium channel gene of Drosophila (Shaker) mutations in a new gene called inebriated (ine) have been isolated. In a wildtype background, ine mutants display no observable behavioral defects. However, in a Sh mutant background, ine mutations cause downturned wings and an indented thorax. This distinctive phenotype is also exhibited by flies of other genotypes that cause extreme neuronal hyperexcitability. The potassium channel blocking drugs quinidine and dideoxy forskolin (DDF) were used to test the effects of ine on synaptic transmission. DDF and ine mutations each potentiate the effects of quinidine on synaptic transmission, but neither have any observable effects in the absence of quinidine. Application of DDF to ine mutants has no effects either in the presence or absence of quinidine. It is concluded that ine mutations increase neuronal membrane excitability (Stern, 1992).

Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. Genetic methods have been used to demonstrate that the Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. Growth of the perineurial glia is controlled by interactions among five genes: ine, which encodes a putative neurotransmitter transporter; eag, which encodes a potassium channel; push, which encodes a large, Zn(2+)-finger-containing protein; amn, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide; and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. These results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis (Yager, 2001).

Mutations in two genes that affect neuronal excitability also affect the structure of the peripheral nerve: double mutants defective in ine, and push exhibit an extremely thickened nerve, which is a phenotype that is clearly visible with the dissecting microscope. To understand the cellular basis for this phenotype, transmission electron microscopy was performed on cross-sections of peripheral nerves. This analysis demonstrated that the push¹ and ine¹;push¹ double mutants exhibit a normal axon and peripheral glial layer, but a thickened perineurial glial layer. This increased perineurial thickness is expressed only moderately in push¹ but very strongly in the ine¹;push¹ double mutant. This increase in thickness is accompanied by an increase in the number of mitochondria within perineurial glial thin sections, suggesting that an increase in cell material accompanies this increased thickness. The ine¹;push¹ phenotype is significantly rescued in transgenic larvae expressing the 943-aa Ine isoform, called Ine-P1, under the transcriptional control of the heat-shock promoter. In particular, perineurial glial thickness in ine¹ push¹; hs-ine-P1 larvae, even in the absence of heat shock, was reduced to 2.0 ± 0.2 µm from 3.1 ± 0.3 in ine¹;push¹. The observed synergistic interaction between ine and push mutations suggests that each gene controls perineurial glial growth through partially redundant pathways (Yager, 2001).

In certain respects, mutations in ine confer phenotypes similar to mutations in the K⁺ channel structural gene eag. In particular, both eag and ine mutations interact synergistically with mutations in the K⁺ channel encoded by Shaker to cause a characteristic 'indented thorax and down-turned wings' phenotype, which is not exhibited by any of the single mutants. Because of this phenotypic similarity, the possibility that eag mutations might also affect perineurial glial thickness was tested. eag¹ resembles ine¹ in the control of perineurial glial growth: eag¹;push¹ double mutants, but not the eag¹ single mutant, exhibit strongly potentiated perineurial glial growth. This increased growth is similar to, but less extreme than, what is observed in ine¹;push¹. Double mutants for eag¹; push² also exhibit a thickened perineurial glial layer. In contrast, eag and ine mutations fail to display a comparable synergistic interaction (Yager, 2001).

Although loss of function mutations in ine confer several phenotypes (Wu, 1977; Stern, 1992; Burg, 1996; X. Huang, 2002), this study (Y. Huang, 2002) focused on two phenotypes that result from increased neuronal excitability. The first phenotype is exhibited by double mutants defective in both ine and the potassium channel alpha subunit encoded by Shaker (Sh). These Sh;ine double mutants exhibit a characteristic 'downturned wings and indented thorax' appearance, which is not exhibited by wild type, or the Sh or ine single mutants (Stern, 1992). This appearance is identical to the appearance of Sh mutants carrying either a mutation in eag, which encodes a potassium channel alpha subunit distinct from Sh, or a duplication of the para gene (termed Dp para⁺), which encodes a Drosophila sodium channel. Because eag mutations and Dp para⁺ each increase neuronal excitability, it has been suggested that this abnormal appearance results when the increased neuronal excitability of Sh mutants is even further increased by a second excitability mutation. The observation that ine mutations confer the identical phenotype suggests that ine mutations increase neuronal excitability as well, perhaps by either increasing sodium currents or reducing potassium currents. The mechanism by which the downturned wings phenotype is elicited by increased neuronal excitability is not known. However, the phenotype might result from hypercontraction of the dorsal longitudinal flight muscles (DLMs), which serve as wing depressors during flight and underlie the area of indented cuticle, as a result of increased neurotransmitter release from the motor neurons (Y. Huang, 2002).

Mutants defective in ine show a second neuronal excitability phenotype, which is manifested at the third instar larval NMJs. Wild-type Drosophila larval NMJs exhibit a phenomenon variously termed long-term facilitation or augmentation. Long-term facilitation occurs after repetitive stimulation of the motor neuron at frequencies such as 5-10 Hz. At some point during this stimulation train, an excitability threshold is reached, and subsequent nerve stimulations then elicit motor nerve depolarizations that are more prolonged in duration, which causes increased Ca²⁺ influx, increased transmitter release, and an increase in the amplitude of the muscle EJP. Certain mutants that exhibit increased neuronal excitability also exhibit an increased rate of onset of long-term facilitation. These mutants include loss of function mutations in Hyperkinetic (Hk), which encodes a K⁺ channel ß subunit, overexpressors of frequenin (frq), which encodes an inhibitor of a K⁺ channel, and Dp para⁺. The observation that ine mutations also increase the rate of onset of long-term facilitation provides further evidence that ine mutations increase neuronal excitability by either increasing sodium currents or reducing potassium currents (Y. Huang, 2002).

Neurotransmitters can affect the properties of target neurons in an acute, short-term manner, or in a long-term manner often involving changes in gene expression. The hyperexcitable phenotype exhibited by ine mutants could be a consequence of chronic overstimulation of the target neurons with the substrate neurotransmitter of Ine during development, leading to long-term increases in neuronal excitability. This effect could require changes in gene expression. Alternatively, the ine mutations could affect neuronal excitability in an acute, short-term manner (minutes to a few hours), which would not be expected to require changes in gene expression. To distinguish between these two possibilities, the ability of Ine-P1 to rescue the downturned wings phenotype of Sh;ine double mutants was investigated when induced transcriptionally during particular times of development. To accomplish this goal, the ine-RA cDNA was introduced into Sh;ine mutants under the control of a heat shock inducible promoter. Transcription of the ine gene was induced by heat shock at various times during development, and the ability to rescue the downturned wings phenotype of Sh;ine double mutants was tested (Y. Huang, 2002).

Whether induction of Ine-P1 expression immediately before eclosion is sufficient for rescue was investigated. Rescue of the downturned wings phenotype occurred when flies carrying the hs-ine-RA were given only one single heat pulse immediately before eclosion. These results suggest that ine expression is not required significantly before eclosion to control the downturned wings phenotype. Induction of Ine-P1 expression after eclosion does not rescue the downturned wings phenotype. The failure of rescue after eclosion perhaps occurs because after eclosion, DLM anatomy is fixed and no longer responds with structural changes to the reduced excitability conferred by ine-RA expression. These results indicate that ine is not required before the time of eclosion to effect the downturned wings phenotype (Y. Huang, 2002).

Neurotransmitters can control the excitability of a target neuron either in a rapid, and rapidly reversible manner or in a long-term manner, often involving changes in gene expression. For example, at the Aplysia sensorimotor synapse, 5-HT application can affect the sensory neuron in both a short-term and long-term manner. In the short term, 5-HT application causes increased excitability of the sensory neuron by cAMP-dependent inhibition of a potassium channel. Long-term exposure, in turn, leads to activation of gene expression by the CREB transcription factor. This study has shown that one aspect of the neuronal excitability phenotype of ine mutants, the 'downturned wings' phenotype of Sh;ine double mutants, can be reverted by a single pulse of ine expression induced immediately before eclosion. This result suggests that Ine is required only in the short term to restore this particular phenotype. Furthermore, this result suggests that any long-term changes in nervous system development that might occur in ine mutants are not sufficient to confer the downturned wings hyperexcitable phenotype. This result further implies that one or more of the ine mutant electrophysiological defects also results from lack of Ine in the short term (Y. Huang, 2002).

It has been proposed that loss of ine function results in defective reuptake of a neurotransmitter, and thus to increased persistence of the transmitter in the synaptic cleft. This increased persistence, in turn, was proposed to cause overstimulation of signaling pathways that would ultimately increase motor neuron excitability (Soehnge, 1996). If so, then it would be predicted that overexpression of Ine might confer the opposite effect: a more rapid clearance of the transmitter, reduced stimulation of signaling pathways controlling excitability, ultimately leading to reduced neuronal excitability. To test this hypothesis, Ine-P1 was overexpressed by crossing the GAL4 drivers MZ1580 or gli-GAL4 to UAS-ine-RA in an otherwise wild-type background. For convenience, overexpression of Ine-P1 will be denoted Overine⁺ in the following discussion (Y. Huang, 2002).

ine mutations enhance the phenotype of Sh mutants, leading to a downturned wings phenotype (Stern, 1992). Overine⁺ confers the opposite phenotype: suppression of the hyperexcitability phenotype of Sh mutants. In particular, whereas Sh mutants shake their legs vigorously after ether anesthesia, Sh MZ1580;UAS-ineRA flies exhibit greatly reduced leg shaking. The reciprocal interactions of ine^- and Overine⁺ with the Sh mutation are consistent with observations in which it was found that hyperexcitability mutations, such as eag^-and Dp para⁺, enhance the phenotypes of Sh mutants, whereas mutations that reduce excitability, such as para loss of function mutations, suppress Sh phenotypes (Y. Huang, 2002). Overine⁺ also confers reduced excitability of the larval motor neuron. In contrast to the increased rate of onset of long-term facilitation observed in ine mutants, Overine⁺ larvae show a decreased rate of onset of long-term facilitation. For example, whereas wild-type larvae required only 2.9 sec of 10 Hz nerve stimulation to induce long-term facilitation, Overine⁺ larvae required 8.4 sec. Similarly, whereas wild-type larvae required only 4.5 sec of 7 Hz stimulation to induce long-term facilitation, Overine⁺ larvae required 24.2 sec. Finally, most of the Overine⁺ larvae failed to exhibit long-term facilitation even after 90 sec of 5 Hz stimulation, whereas most wild-type larvae were able to induce long-term facilitation under these conditions (Y. Huang, 2002).

The decreased neuronal excitability observed in Overine⁺ larvae could be a consequence of decreased sodium channel activity. Mutants with decreased sodium channel activity, such as para, tipE, mle^nap, Kinesin heavy chain, and axotactin often show a ts paralytic phenotype. These mutants, but not wild type, become paralyzed very quickly (within seconds or minutes) after placement at the elevated temperature, which can range from ~29° to 38°. Generally, more severe reductions in sodium currents lead to a reduction of the temperature required to induce the paralysis. Overine⁺ also confers ts paralysis. In particular, 92% of flies carrying both the UAS-ine-RA and the gli-gal4 constructs became paralyzed after transfer from 18° to 38°, whereas flies carrying only the UAS-ine-RA construct or only the gli-gal4 construct did not show this paralysis (Y. Huang, 2002).

Mutations affecting neuronal excitability often display synergistic interactions. Because Overine⁺ and some para mutations cause ts paralysis, a possible synergistic interaction between the two was tested. In particular, assays were carried out for ts paralysis in flies combined for Overine⁺ and para⁶³, which is a partial loss of function mutation in para that confers ts paralysis. Although almost all para⁶³ and Overine⁺ mutants become paralyzed at 38°, only 2.2% of the para⁶³ flies and 4.5% of the Overine⁺ flies became paralyzed when placed at 29°C. However, when the gli-gal4 and UAS-ine-RA constructs were cointroduced into the para⁶³ background, to form the Overine⁺ para⁶³ combination, 73% of the flies became paralyzed at 29°C. Furthermore, whereas only 6.7% of the para⁶³ flies and 18% of the Overine⁺ single mutant became paralyzed, respectively, when placed at 32°C, all of the para⁶³;Overine⁺ double mutants tested became paralyzed. This result demonstrates that strong synergistic enhancement occurs between Overine⁺ and para⁶³ (Y. Huang, 2002).

The resemblance of Overine⁺ to para⁶³ is manifested not only at the behavioral level but also at the electrophysiological level. Compared with wild type, both para⁶³ and Overine⁺ larvae exhibit a higher frequency of failures in evoked transmitter release from larval motor nerve terminals when bathed in buffer containing low [Ca²⁺]. This phenotype reflects a presynaptic defect: the amplitude of miniature EJPs (mEJPs) is unchanged by Overine⁺ or para⁶³. Furthermore, the amplitude of successful EJPs is unaffected in Overine⁺ or para⁶³ larvae at the lowest [Ca²⁺] tested (0.1 mM), for which only failures or releases of single vesicles occurs. This increased failure rate is likely to result from an axonal action potential of attenuated amplitude, which reduces the consequent nerve terminal Ca²⁺ influx, and thus reduces the probability of synaptic vesicle release. This interpretation predicts that Overine⁺ or para⁶³ should shift the Ca²⁺/transmitter release curve to the right, which is in fact what is observed (Y. Huang, 2002).

Further evidence for an attenuated action potential amplitude in Overine⁺ or para⁶³ was obtained from extracellular recordings of compound action potentials of the motor and sensory axons of the segmental nerve. The compound action potential is the additive output of action potentials fired by each axon in the nerve bundle in response to nerve stimulation. At the permissive temperature of 21°-22°C, both Overine⁺ flies and para⁶³ larvae show compound action potential of reduced amplitude compared with wild type. Furthermore, at the restrictive temperature of 38°, at which both Overine⁺ and para⁶³ adults exhibit paralysis, both Overine⁺ and para⁶³ larvae showed complete loss of compound action potentials. The loss is reversed when the temperature is lowered to the permissive temperature. Temperature-sensitive loss of action potential propagation has been reported for other mutants showing reduced sodium currents as well. This loss of action potentials is presumably related to the temperature-sensitive paralytic phenotype that these mutants exhibit. Compound action potentials of reduced amplitude at the permissive temperature is also a feature of mutants defective in Khc, which encodes kinesin heavy chain: this phenotype was suggested to result at least in part from a reduction in axonal sodium channels as a consequence of defective axonal transport. Taken together, these results suggest that overexpression of Ine-P1 reduces sodium channel activity and that the substrate neurotransmitter of the Ine transporter might control a signaling pathway that ultimately targets sodium channels (Y. Huang, 2002).

Three possible mechanisms are suggested to account for these data. (1) The substrate transmitter of Ine is released from an interneuron that synapses onto the motor neuron. Binding of the transmitter to its receptors in the motor neuron triggers a signal transduction pathway that serves to activate sodium channels in the motor neuron. The Ine transporter, which resides either in the interneuron, the motor neuron, or a neighboring glia, terminates this signaling pathway. In preparations for electrophysiology recordings, the motor neuron cell body, together with any upstream interneurons, are severed from the axon and removed. If the substrate neurotransmitter of Ine is released from the interneuron then it must exert its effects on motor neuron excitability before the dissection. This possibility does not necessarily contradict the hypothesis that Ine affects excitability in a short-term manner, because there are several molecular mechanisms that can operate on the required time scale. For example, CAM kinase II autophosphorylation causes its signaling pathway to remain active for a prolonged period, even in the absence of the original stimulus (Y. Huang, 2002).

(2) The substrate neurotransmitter of Ine is released from the motor nerve terminal and acts on autoreceptors on the motor neuron. In this model, Ine could function from either the motor neuron or the peripheral glia to terminate this signaling. As above, binding of the transmitter to its receptors in the motor neuron triggers a signal transduction pathway that activates sodium channels. Sodium channels near the nerve terminal would be the most prominent candidates for this activation. However, the reduced axonal action potential amplitudes observed in Overine⁺ would require that the signal be transduced from the motor nerve terminal along the length of the axon (Y. Huang, 2002).

(3) The substrate neurotransmitter of Ine is released from the motor neuron and activates receptors in the peripheral glia. The activated peripheral glia then release factors that act reciprocally on the motor axon to increase sodium currents, thus forming a positive feedback loop. It is well documented that neurons release factors that affect adjoining glia and that glia can produce factors that increase neuronal excitability. For example, at the frog neuromuscular junction, motor nerve stimulation or neurotransmitter application increase intracellular [Ca²⁺] in perisynaptic Schwann cells. Glial also release substances that affect excitability of the neurons. For example, the Drosophila axotactin< (axo) gene encodes a neurexin-related protein that is produced by peripheral glia and subsequently localized to axon tracts. Mutations in axo cause temperature-sensitive paralysis and failure of compound action potentials at the restrictive temperature: these are phenotypes exhibited by Overine⁺ larvae as well and they presumably result from reductions in axonal sodium currents. This mechanism requires that production or release of this excitability factor from peripheral glia be increased in ine mutants and reduced in Overine⁺ larvae. Yager (2001) has proposed that ine mutations increase the release of a factor from peripheral glia that increases the growth of the outer perineurial glial layer. This proposal is consistent with the mechanism proposed in this study (Y. Huang, 2002).

Two observations raise the possibility that ine might be required for osmolyte transport and thus for the Drosophila osmotic stress response. (1) Both forms of ine are expressed robustly in fluid reabsorption tissues such as the Malpighian tubule, hindgut, and anal plate (Soehnge, 1996), which together comprise the invertebrate analog of the kidney; (2) transport of the osmolytes betaine, taurine, and ß-alanine into cells in the mammalian renal medulla is accomplished by transporters such as BGT1 that are members of the same transporter family as ine. These observations raised the possibility that the Ine transporter might function to transport osmolytes as well (X. Huang, 2002).

If Ine performs osmolyte transport in the Malpighian tubules and hindgut, then ine mutants, which would be defective in such transport, would be expected to be more sensitive to osmotic stress than wild-type flies. To test this possibility, three independently isolated ine mutants and wild-type flies were maintained on media containing various [NaCl]. ine mutants exhibit viability similar to wild type when maintained for 4 days on 0 M or 0.1 M [NaCl]. However, ine¹ and ine³ mutants exhibit significantly greater lethality than wild-type or ine² mutants when maintained for 4 days on 0.2 M [NaCl]. Furthermore, whereas ~90% of wild-type flies can survive maintenance on 0.4 M [NaCl], ine¹ and ine³ mutants exhibit essentially complete inviability on this [NaCl], and ine² mutants exhibited only slight viability. The abdomens of both wild-type and ine mutants become progressively thinner during their maintenance on lethal, but not sublethal, [NaCl]. This observation is consistent with the possibility of desiccation, which might contribute to the observed lethality (X. Huang, 2002).

To confirm that this reduced viability reflects increased sensitivity to a hypertonic medium, rather than increased sensitivity specific to NaCl, the sensitivity of ine mutants to elevated [KCl] and [sorbitol] was tested. ine mutants display increased sensitivity to both, although the sensitivity of both wild-type and ine mutants to sorbitol is considerably less than the sensitivity to NaCl and KCl. This significantly reduced sensitivity to sorbitol compared to NaCl and KCl suggests that the observed lethality in NaCl and KCl might not arise solely from desiccation. One possibility is that some of the NaCl and KCl provided to the flies might accumulate intracellularly and contribute to lethality. Alternatively, the reduced sensitivity to sorbitol might result from some ability of sorbitol to cross the cell membrane, which would give sorbitol a partial osmoprotective effect. As with NaCl, ine² mutants exhibited slightly better survival than ine¹ and ine³ mutants when maintained on media containing 0.2 M [KCl], although the difference is less extreme than the difference observed on NaCl-containing media (X. Huang, 2002).

To test the possibility that ine mutants might be hypersensitive to any environmental stress, rather than specifically sensitive to hypertonic stress, the sensitivity of ine³ flies and wild type were compared to two types of heat-shock stresses: long-term maintenance at a temperature of 34° and 3-hr heat shocks at 37° during long-term maintenance at room temperature. ine³ flies display the same viability as wild type to these stresses (X. Huang, 2002).

The phenotype of ine¹ and ine³ mutants most likely represents the null phenotype: ine³ is a deletion mutation that removes most of the ine open reading frame, and ine¹ mutants produce undetectable levels of mRNA from either of the ine isoforms (Soehnge, 1996), although the ine¹ sequence change was not identified. The observation that ine² mutants survive significantly better than ine¹ and ine³ mutants on media containing 0.2 M NaCl or 0.2 M KCl suggested that the ine² mutation does not completely eliminate Ine activity. To identify the ine² mutation, the sequences of ine in the ine² mutant and in the isogenic wild-type strain were compared. ine² is a nonsense mutation in codon 125 of the Ine-P1 isoform. This mutation is expected to eliminate Ine-P1, but since this mutation lies in an exon that is not present in the Ine-P2 isoform, it is expected to leave Ine-P2 unaffected. The observation that the ine² mutant retains partial activity for the osmotic stress response demonstrates that Ine-P1 is required for most of, but not all of, the osmotic stress response. Ine-P2 alone is sufficient for a small amount of osmotic stress response (X. Huang, 2002).

An additional way to assess the role of each ine isoform is to assay the osmotic stress response in transgenic flies carrying each isoform independently. ine mutants expressing ine-RA under transcriptional control of the heat-shock promoter completely rescues the increased sensitivity of ine mutants to NaCl, even in the absence of heat shock. In addition, flies carrying ine-RB under the transcriptional control of the upstream activator sequence of the yeast Gal4 protein (UAS-ine-RB) were constructed. ine mutants are completely rescued for the phenotype of NaCl sensitivity in the simultaneous presence of UAS-ine-RB and a transgene ubiquitously expressing GAL4 (called hs-GAL4. In contrast, ine mutants expressing either the hs-GAL4 line or the UAS-ine-RB line alone exhibit an identical sensitivity to NaCl as ine mutants. Thus, expression of Ine-P2, via the GAL4 system, but not expression of Ine-P2 from its normal chromosomal position, is sufficient for a normal osmotic stress response even in the absence of Ine-P1. It is suggested that this ability of Ine-P2 to rescue is a result of its overexpression by the GAL4 system, although this overexpression has not been demonstrated (X. Huang, 2002).

ine mutants, but not wild-type flies, die following maintenance on media containing 0.2 or 0.4 M NaCl. However, because these data represent viability at only a single time point, no information on mortality kinetics were obtained. The rate of death of ine¹, ine², and wild-type flies on media containing varying [NaCl] was compared. Each genotype exhibits a 'threshold' [NaCl]: flies maintained on media containing [NaCl] below the threshold exhibit very little lethality, even after 9 days of maintenance on the hypertonic medium. However, flies maintained on media containing [NaCl] above the threshold died quickly (death typically began within 3–5 days following addition to the hypertonic media) and continuously until, after 9 days upon NaCl-containing media, <10% of the flies remained alive. The [NaCl] at which this threshold response occurs depends on the allele present at ine. Wild-type flies exhibit an [NaCl] viability threshold between 0.5 and 0.6 M [NaCl]. In contrast, ine¹ mutant flies exhibit a [NaCl] viability threshold between 0.15 and 0.2 M [NaCl]. Finally, ine² mutants exhibit a threshold concentration between 0.2 and 0.25 M [NaCl], which is intermediate between wild type and ine¹ and mutants. Thus, there is a close correlation between the strength of the mutant allele at ine and the sensitivity of the fly to osmotic stress. This observation suggests that threshold [NaCl] is determined, at least in part, by the amount of osmolyte accumulation that can be performed in the Malpighian tubule and hindgut (X. Huang, 2002).

The role of carcinine in signaling at the Drosophila photoreceptor synapse

The Drosophila photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized Inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. By feeding wild-type flies carcinine, components of mutant ine ERGs can be duplicated. Finally, it was demonstrated that treatment with H₃ receptor agonists (H₃ receptor is a presynaptic G-protein-coupled autoreceptor, a metabotropic histamine receptor, that inhibits histamine release) or inverse agonists rescue several components of the mutant ine ERG phenotype. This sutdy provides pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. It is also speculated that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H₃ receptor (Gavin, 2007).

The findings of this study indicate that the presumed neurotransmitter transporter encoded by the ine gene in Drosophila transports the histamine metabolite carcinine. Using genetic epistasis this study shows that the oscillations observed in mutant ine ERGs require histidine decarboxylase activity and the carcinine-synthesizing enzyme Ebony, but not the carcinine-hydrolyzing enzyme Tan. Treating wild-type flies with carcinine can phenocopy components of the mutant ine ERG phenotype. Finally, by rescuing the ine²-associated phenotype with drugs that target the mammalian H₃ receptor, pharmacological evidence is provided for the presence of a yet uncharacterized putative H₃ receptor in Drosophila that may be responsible for the ERG oscillations observed in flies carrying mutations in the ine gene (Gavin, 2007).

Previous studies involving intracellular voltage recordings of ine mutants have led to the conclusion that the oscillations observed in ine mutant ERGs are the result of a defect occurring within the photoreceptor cell. These conclusions are supported by expressing ine specifically in photoreceptor cells and demonstrating a rescue of the ine²-associated oscillations. Neurotransmitter transporters are often able to function from either the presynaptic neuron or from neighboring glial cells, as shown at the neuromuscular junction in ine mutants. Glial cell-specific expression of the ine gene in ine² flies results in a complete rescue of the ine mutant ERG phenotype. It was somewhat unexpected that ine expression in glial cells rescued the ine² phenotypes, since glial cells have been shown to lack Tan protein and thus would be unable to convert carcinine back to a recycled pool of histamine. However, it is possible that glial cells do express trace amounts of the enzyme Tan to hydrolyze carcinine and generate a renewable source of histamine for photoreceptor cells, and it is also possible that the Inebriated protein is expressed in a non-autonomous manner and can be transported from glial cells to photoreceptors in the fly eye (Gavin, 2007).

The finding that an ERG recording can exhibit oscillations is somewhat surprising. An ERG does not record the electrical response of a single photoreceptor, but rather is a collective measure of the retinal photoresponse. Thus, if the mutant ine-associated ERG defects are indeed localized to the photoreceptor synapse, as the data suggest, then one would expect that different photoreceptors would be excited/inhibited at different timepoints, ultimately resulting in the oscillations simply canceling themselves out. The fact that oscillations are indeed observed, and appear to be due to a defect occurring at the photoreceptor synapse, implies the existence of an uncharacterized and complex synchronization of photoreceptor cell de-/repolarization (Gavin, 2007).

The lack of rescue of ine²-associated oscillations in flies carrying additional mutations in the postsynaptic histamine receptor gene ort, the finding that mutant ine oscillations were detected within single photoreceptor cells, and the observations that the mutant ine phenotype can be rescued when ine is expressed in photoreceptors, all combine to strongly suggest that the oscillation phenotype is likely a result of a defect occurring within the photoreceptor itself. In addition, by crossing ine² animals with Hdc^P218 flies, it was demonstrated that the ine²-associated oscillations are dependent upon histamine synthesis. All of these results indicate that histamine is somehow contributing to the aberrant ERG witnessed in ine² flies, and that histamine appears to be acting on the presynaptic photoreceptor cell to induce this oscillation phenotype. Further epistatic analyses also revealed that Ebony, but not Tan, activity is required for the generation of oscillations in ine² ERGs. These genetic experiments are consistent with ine encoding either a carcinine importer found in the photoreceptor cell or a carcinine exporter found in glial cells. The homology of Inebriated with other known Na⁺/Cl^- neurotransmitter transporters (which import neurotransmitter into cells) suggests that Inebriated protein is transporting carcinine into the photoreceptor, and not out of glial cells (Gavin, 2007).

While Ebony is known to act on multiple substrates, such as dopamine to generate β-alanyl-dopamine, the requirement of histamine synthesis for the maintenance of ine²-associated oscillations suggests that it is β-alanyl-histamine, or carcinine, that is somehow responsible for the oscillations observed in ine² ERGs. It should be noted, however, that ebony mutations were not sufficient in rescuing the hyperpolarization response observed in mutant ine ERG traces. The origins of this hyperpolarization response are still unclear and further research will be required to elucidate its exact meaning. In tan mutants, one would predict that there would be a buildup of carcinine. However, this buildup does not give rise to an ERG recording similar to that of ine². This is due most likely to the presence of functional Inebriated protein in tan mutant flies, which should effectively clear the carcinine from the synaptic cleft for degradation within the photoreceptor cell (Gavin, 2007).

By treating wild-type and ebony¹¹ flies with carcinine and subsequently inducing components of the ine²-ERG phenotype, further evidence is provided suggesting that the sharp depolarization spike, the oscillations, and the hyperpolarization response all seen in ine²-ERGs are due to a buildup of carcinine within the photoreceptor synaptic cleft. While the oscillations observed in carcinine-treated wild-type flies do not mimic exactly the oscillations seen in ine² ERG recordings, it is presumably difficult to replicate the carcinine and histamine balance occurring in the eyes of ine² animals. Indeed, treatment of wild-type flies with higher (10%) or lower (1%) concentrations of carcinine were less effective in inducing the oscillations than the described 5% carcinine dose (Gavin, 2007).

It is possible that carcinine is being degraded or modified by the fly before the compound is able to exert its effects at the photoreceptor cell. In order to eliminate the activity of one enzyme known to be involved in carcinine metabolism, tan¹ flies were treated with 5% carcinine overnight. Surprisingly, none of the tan¹ flies treated with carcinine showed an aberrant ERG phenotype. It was surprising that carcinine treatment had a strong effect in flies of the ebony¹¹, but not the tan¹, background. While the results of these tan¹ and ebony¹¹ carcinine-treatment experiments are unexpected, one possible explanation may involve the regulation of carcinine clearance/degradation. The tan¹ flies presumably suffer from a perpetual excess of carcinine even before exogenous carcinine treatment, and these flies, in order to reduce their sensitivity to this compound, may consequently decrease the levels of a putative carcinine receptor, increase their rate of carcinine degradation, or increase the levels of Inebriated protein for carcinine clearance. However, ebony¹¹ flies are relatively 'naive' to the effects of carcinine, as their ability to synthesize this compound has been greatly diminished, and as a result these animals may have an increased level of the supposed carcinine receptor, a decrease in Inebriated receptor levels or a decrease in carcinine degradation, ultimately making them more sensitive to the effects of carcinine treatment (Gavin, 2007).

It remains to be seen whether or not all of the mutant ine-associated phenotypes, including increased neuronal excitability and increased sensitivity to osmotic stress, are due to the inability of these flies to transport carcinine. It is possible that the Inebriated protein transports other compounds that perhaps share the common feature of β-alanine conjugation. This might help explain why none of the more common neurotransmitters were taken up by ine-transfected Xenopus oocytes. In order to assist in confirming that Inebriated is indeed a carcinine neurotransmitter transporter, in vitro experiments, such as neurotransmitter uptake assays, will need to be performed. In addition, the ability of Inebriated protein to take up other β-alanyl-neurotransmitters/osmolytes also should be examined (Gavin, 2007).

The oscillations present within the photoreceptor response of ine² ERGs appear as sharp depolarization/repolarization spikes, and this oscillation phenotype is dependent upon both histamine synthesis and Ebony activity, and is sensitive to drugs that target mammalian H₃ receptors. It is perplexing that the synthesis of a single metabolite, carcinine, could be responsible for both the depolarization and repolarization spikes observed within ine mutant ERGs. It is speculated that these oscillations are the result of aberrant signaling involving both carcinine and histamine at a putative H₃ receptor in Drosophila. H₃ receptors are an unusual example of the G-protein coupled receptor family, in that they have partial constitutive activity, resulting in a constant small percentage of stimulated G-proteins that trigger a reduction of histamine synthesis and release as well as a decrease in extracellular calcium inflow. The presence of an H₃ receptor agonist, such as histamine, causes an increase in activity of the associated G-protein and therefore a stronger inhibition of both histamine release and calcium inflow. Thus, synaptic histamine serves as a negative regulator for its own release and induces a slight repolarization of a stimulated presynaptic histaminergic neuron by inhibiting presynaptic calcium channels. An H₃ receptor inverse agonist is believed to act by blocking the constitutive activity of the H₃ receptor, resulting in the liberation from a histamine release checkpoint as well as the release of restrictions on calcium inflow. Recently, it has been shown that carcinine has the ability to act as an inverse agonist of presynaptic H₃ receptors in mice. While significant further research is required to confirm this hypothesis, it is surmised that histamine and carcinine are exerting opposing effects on the polarization state of the histaminergic photoreceptor cell by activating or inhibiting presynaptic calcium channels via a putative Drosophila H₃ receptor. While a recent search of the Drosophila genome did not uncover any direct homologs to vertebrate metabotropic histamine receptors, the CG7918 gene was listed as a possible candidate for encoding such a receptor, and this gene bears strong homology to genes encoding H₃ receptors in mammals. In addition, the ine²-associated oscillations display sensitivity to mammalian H₃ receptor agonists and inverse agonists, strengthening the possibility that an H₃ receptor does exist in Drosophila. It is still unclear what the origins of the thioperamide-sensitive depolarization spikes are that are observed in ort⁵ ERGs. The presence of these thioperamide-sensitive spikes in ort⁵ ERG recordings implies the requirement of some postsynaptic retrograde signal for ERG stability, and this ort-dependent signal may be involved in the sensitization of the putative H₃ receptor (Gavin, 2007).

It was unexpected that thioperamide treatment of wild-type flies resulted in the loss of on and off transients within their ERG traces. It is possible that histamine release was so extreme in the presence of the potent thioperamide that histamine levels were nearly depleted in the eye, resulting in the disruption of downstream signaling events. Indeed, treatment of mice with high concentrations of carcinine, which acts as an inverse agonist of H₃ receptors similar to thioperamide, was shown to result in significantly lower overall levels of histamine within the brains of treated mice. This model of indirect histamine depletion has also been postulated to occur in ebony mutant flies. The absence of on and off transients in ebony mutant ERG recordings is attributed to the normal release of histamine by photoreceptor cells, but this histamine subsequently lacks the ability to be 'trapped' by β-alanine conjugation, ultimately resulting in histamine diffusing away from the eye. Interestingly, expression of pertussis toxin in photoreceptor and laminar neurons in Drosophila results in a similar loss of on and off transients in ERG traces, and this is believed to be the result of inactivation of an unknown G-protein coupled receptor found in photoreceptor cells that is unlikely to be rhodopsin. It is possible that pertussis toxin was acting within photoreceptor cells upon the putative H₃ receptor in this study, resulting in a lack of negative feedback on histamine synthesis/release, eventually causing the exhaustion/depletion of histamine pools. Further research will be required to confirm or dismiss the presence of a histamine/carcinine-sensitive H₃ receptor in Drosophila photoreceptor cells (Gavin, 2007).

REFERENCES

Search PubMed for articles about Drosophila inebriated

Burg, M. G., Geng, C., Guan, Y., Koliantz, G. and Pak, W. L. (1996). Drosophila rosA gene, which when mutant causes aberrant photoreceptor oscillation, encodes a novel neurotransmitter transporter homologue. J. Neurogenet. 11: 59-79. 10876650

Chiu, C., Ross, L. S., Cohen, B. N., Lester, H. A. and Gill, S. S. (2000). The transporter-like protein inebriated mediates hyperosmotic stimuli through intracellular signaling. J. Exp. Biol. 203 Pt 23: 3531-46. 11060215

Gavin, B. A., Arruda, S. E. and Dolph, P. J. (2007). The role of carcinine in signaling at the Drosophila photoreceptor synapse. PLoS Genet. 3(12): e206. PubMed Citation: 18069895

Huang, X., (2002). The Drosophila inebriated-encoded neurotransmitter/osmolyte transporter: dual roles in the control of neuronal excitability and the osmotic stress response. Genetics 160: 561-569. 11861562

Huang, Y. and Stern, M. (2002). In vivo properties of the Drosophila inebriated-encoded neurotransmitter transporter. J. Neurosci. 22(5): 1698-1708. 11880499

Luan, Z., Quigley, C. and Li, H. S. (2015). The putative Na(+)/Cl(-)-dependent neurotransmitter/osmolyte transporter Inebriated in the Drosophila hindgut is essential for the maintenance of systemic water homeostasis. Sci Rep 5: 7993. PubMed. PubMed ID: 25613130

Soehnge, H., Huang, X., Becker, M., Whitley, P., Conover, D. and Stern, M. (1996). A neurotransmitter transporter encoded by the Drosophila inebriated gene. Proc. Natl. Acad. Sci. 93: 13262-13267. 8917579

Stern, M. and Ganetzky, B. (1992). Identification and characterization of inebriated, a gene affecting neuronal excitability in Drosophila. J. Neurogenet. 8: 157-172. 1334137

Wu, C.-F. and Wong, F. (1977). Frequency characteristics in the visual system of Drosophila: genetic dissection of electroretinogram components. J. Gen. Physiol. 69: 705-724. 894240

Yager, J., Richards, S., Hekmat-Scafe, D. S., Hurd, D. D., Sundaresan, V., Caprette, D. R., Saxton, W. M., Carlson, J. R. and Stern, M. (2001). Control of Drosophila perineurial glial growth by interacting neurotransmitter-mediated signaling pathways. Proc. Natl. Acad. Sci. 98: 10445-10450. 11517334

date revised: 25 March 2015

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