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).

GENE STRUCTURE

cDNA clone length - 3588 (Ine-P1 isoform)

Bases in 5' UTR - 621

Exons - 10

Bases in 3' UTR - 1242

PROTEIN STRUCTURE

Amino Acids - 943 (Ine-P1) and 658 (Ine-P2)

Structural Domains

Mutations in ine were localized on cloned DNA by restriction mapping and restriction fragment length polymorphism (RFLP) mapping of ine mutants. DNA from the ine region was then used to isolate an ine cDNA. The ine cDNA contains an open reading frame of 658 amino acids (Ine-P2) with a high degree of sequence similarity to members of the Na+/Cl(-)-dependent neurotransmitter transporter family. Members of this family catalyze the rapid reuptake of neurotransmitters released into the synapse and thereby play key roles in controlling neuronal function (Soehnge, 1996).

Members of the of the Na+/Cl-dependent neurotransmitter transporter family have been shown to catalyze the reuptake of neurotransmitters such as glycine, dopamine, 5HT, NE, GABA, as well as metabolites or osmolytes such as taurine, betaine, ß-alanine, and creatine. The Ine transporter shows the strongest sequence similarity to the rat GABA transporter and human GABA/betaine transporter (40.9% and 40.6% sequence identity, respectively); however, the Ine transporter also shows significant (40.4% and 39.9% identity, respectively) homology to the human dopamine and NE transporters. Several structural features common to members of this family are observed in Ine. These features include 12 transmembrane domains, a large extracellular domain between the third and fourth transmembrane domains that contains two cysteine residues spaced 9 amino acids apart (residues 173 and 182) and a conserved tryptophan (residue 243) in the fourth transmembrane domain that has been proposed to be important for incorporation of members of this family into the membrane. However, the Ine transporter is apparently a more distantly related member of this family: Ine displays differences in 51 of the 168 amino acids conserved among five other transporters. The Ine transporter does not appear to be more closely related to the Drosophila 5HT transporter dSERT than to other transporters. In fact, there are only 19 amino acids that are the same in Ine and dSERT but different in the four other transporters shown. In contrast, there are 31 amino acids that are the same between Ine and the two GABA transporters but different in the three monoamine transporters examined (Soehnge, 1996).

The Drosophila receptor oscillation A (rosA) mutations, which cause electroretinogram (ERG) defects, including oscillations, were localized to the 24F4-25A2 region of chromosome 2L. Genomic fragments from this region, isolated from bacteriophage P1 clones, include those that detect transcriptional defects in rosA mutants in RNA blot experiments. One of these genomic fragments was used to screen a head cDNA library. The largest cDNA clone (3.6 kb) isolated was shown to rescue a rosA mutant in P element-germline transformation experiments. The ROSA protein deduced from the open reading frame in the 3.6 kb rosA cDNA is 943 amino acids long (Ine-P1) and is 36%-41% identical to members of the superfamily of Na+/Cl(-)-dependent neurotransmitter transporters, with no indication of higher sequence identity to any one subgroup within the superfamily. RNA blot experiments have revealed multiple transcripts in various developmental stages, the most abundant one being a 3.7 kb transcript, particularly in the adult head. The results demonstrate that the rosA gene encodes a novel Na+/Cl(-)-dependent transporter important for normal response properties of the photoreceptor (Burg, 1996).

date revised: 11 August 2002

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