See the embryonic expression pattern of ine at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
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).
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, ICl(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 ICl(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 ICl(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).
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, mlenap, 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).
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date revised: 11 October 2002
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