Chemical Senses Vol. 30 No. suppl 1 © Oxford University
Press 2005; all rights reserved
Trehalose Sensitivity of the Gustatory Receptor Neurons Expressing Wild-type, Mutant and Ectopic Gr5a in Drosophila
1 Tohoku University Graduate School of Information Sciences, Sendai 980-8579, Japan, 2 Gunma University School of Medicine, Maebashi 371-8511, Japan and 3 Drosophila Genetic Resource Center, Kyoto Institute of Technology, Kyoto 616-8354, Japan
Correspondence to be sent to: Kunio Isono, e-mail address: isono{at}bio.is.tohoku.ac.jp
Key words: Drosophila, feeding, gustatory receptor, Gr5a, taste sensitivity, trehalose
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Introduction |
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 Top Introduction ResultsDiscussionReferences |
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Among ~70 candidate gustatory receptor genes identified from the Drosophila genome, Gr5a is the only gene that is allelic to a known gene, Tre, which controls taste sensitivity. Tre was discovered as an X-linked genetic polymorphism (Tre+ and Tre01) among wild populations and laboratory strains (Tanimura et al., 1982
).
Recent molecular studies on Gr5a and other gustatory receptor genes supported
the finding that Tre encodes a functional gustatory sugar receptor and have
provided novel information on the sugar sensitivity in the gustatory receptor neurons.
Gr5a was found to be one of the candidate gustatory receptor genes discovered
from the Drosophila genome database (Clyne et al., 2000), with the locus in close
proximity to Tre (Berkeley Drosophila Genome Project;http://flybase.net).
Mutations were obtained using a P-element insertion near the Tre locus
(Isono et al., 1998).
Subsequent molecular analysis of the mutations provided key information to prove that
Tre is identical to Gr5a (Dahanukar et al., 2001;
Ueno et al., 2001). The
induced mutations and the spontaneous mutation Tre01 provide
a clue to the understanding of how a specific chemosensory receptor protein contributes
to the sensitivity of the receptor neurons and the feeding response. In this paper we
present physiological and behavioral data for the gustatory receptor TRE encoded by
Gr5a in wild-type, mutant and transformant flies and discuss the sugar
sensitivity of the gustatory receptor neurons.
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Results |
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TopIntroductionResultsDiscussionReferences |
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The dosage of the receptor TRE and the gustatory sugar sensitivity
A feeding preference test (Ueno et
al., 2001) to various concentrations of trehalose against control 2 mM
sucrose was investigated in a trehalose-sensitive strain Canton-S
(Tre+), an insensitive strain Oregon-R
(Tre01) and the F1 females
(Tre+/Tre01) from the
cross of the two strains (Figure
1A). Trehalose sensitivity was defined
as the concentration of trehalose that gives a preference index of 0.5. The mean
sensitivity was estimated to be 9.8, 54 and 21 mM for Canton-S,
Oregon-R and the F1 females, respectively. Thus the F1 females showed an
intermediate value of the two parents, as was reported previously (Tanimura et al., 1982). We then carried out a
simpler preference test with a fixed concentration of 20 mM trehalose versus 2 mM sucrose
in transformant flies where a 4.6 kb EcoRI–NotI genomic fragment
containing the Gr5a gene or a 7.2 kb HindIII–EcoRI
fragment containing the CG3171 gene was ectopically introduced. A total of 10
independent transformants for each gene was obtained with a host strain
w1118Tre01 and was mapped for
each insertion. The mean preference index, based on nine CG3171 transformants
and nine Gr5a transformants, is shown in Figure
1B. CG3171 transformant flies
did not significantly modify the preference for both hemizygotes and homozygotes for the
insertions, supporting the previous report by
Dahanukar et al. (2001) but
not supporting the result of
Ishimoto et al. (2000), where
ectopic CG3171 rescued induced Tre mutation. In Gr5a
transformants, however, the preference index was noticeably increased as was previously
reported in an experiment where Gr5a was shown to rescue
Tre
EP5 and TreEP19 mutations
(Dahanukar et al., 2001).
Note that ectopic Gr5a homozygotes almost fully rescued
Tre01 on the host X-chromosome. The Gr5a
hemizygotes showed an intermediate sensitivity. Therefore we conclude that the
Gr5a or Tre+ allele positively and
gene-dose-dependently contributes to the trehalose sensitivity regardless of its ectopic
or intrinsic origin. Hemizygous Gr5a transformants carrying homozygous intrinsic
Tre01 showed a similar intermediate trehalose sensitivity as
in Tre+/Tre01 heterozygous
F1 females. Therefore it is not supported that Tre01
negatively contributes to decrease trehalose sensitivity.
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Effect of Tre mutations on the sugar sensitivity of the receptor neurons
Extracellular recordings from the tips of the labellar taste hairs of
Drosophila provide information on neural activity of the unit gustatory receptor
neurons. Stimulation with a sugar solution usually evokes a train of impulses different
from the impulses evoked by water stimulation (Figure
2A;
Fujishiro et al., 1984). The
number of impulses arising from sugar-sensitive neurons from 0.2 to 0.4 s after onset of
the stimulation was compared in two Tre+ and two
Tre mutant strains (Figure
2B): Canton-S
(Tre+), EP(X)496
(Tre+), which is a parent strain used to induce
P-element excision mutations by
Isono et al. (1998),
Oregon-R (Tre01) and a P element excision mutant
EP3 (TreEP3).
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All four strains responded normally to sucrose stimulation (data not shown). Stimulation with trehalose solutions gave different responses depending on Tre alleles: very good responses were obtained to various concentrations of trehalose in the two Tre+ strains (Canton-S and EP(X)496), while the response was noticeably reduced in
EP3, as
was reported by
Dahanukar et al. (2001) for
two EP mutations, TreEP5 and
TreEP19. The responses of EP3 and
Oregon-R, however, were not totally extinguished. Both strains showed similar
trehalose sensitivities at higher trehalose concentrations. By comparing the
concentration–response relationships of the four strains, it was suggested that
5–10 times higher trehalose concentrations are necessary to attain a similar level
of response in Oregon-R and EP3 as compared with the two
Tre+ strains, suggesting a corresponding decrease in
the trehalose sensitivity by the Tre mutations. |
Discussion |
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TopIntroductionResultsDiscussionReferences |
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In the feeding preference test we observed a gene-dose-dependent, positive contribution of Tre+ allele to the gustatory trehalose sensitivity (Figure 1A,B). In the electrophysiological experiment it was also shown that normal gene-dose of intrinsic or ectopic Tre+ ensures trehalose sensitivity of the receptor neurons (Figure 2B; Dahanukar et al., 2001
).
Therefore the amount of the functional gustatory receptor TRE encoded by
Tre+ may be produced gene-dose dependently.
On the other hand, Tre01contribution was not apparently
observed in the feeding preference tests. In the receptor neurons
Tre01did not contribute to the electrophysiological
trehalose sensitivity since Tre01 flies (Oregon-R)
showed a trehalose sensitivity not significantly higher than in EP3,
where the Gr5a gene is severely disrupted and considered to produce no
functional mRNAs and the receptor proteins (Ueno
et al., 2001). Tre01 is a polymorphic
amino residue substitution Thr218Ala in the second intracellular loop domain of the
seven-transmembrane protein TRE (Ueno et
al., 2001). It was also recently shown that, among the polymorphic sites
in Gr5a, Thr218Ala is exclusively involved in controlling the trehalose
sensitivity (Inomata et al.,
2004). Taken together, the present results suggest that
Tre01 is a null mutation that inactivates the receptor
function in an all-or-none fashion. The mutation may abolish binding interaction of the
receptor with a sugar ligand or interaction with G protein and/or its activation. Future
structure–function studies of Tre01 may provide clues
to understanding the molecular mechanism of the receptor function and the gustatory
transduction mechanism.
Where does the residual sensitivity to trehalose observed in Oregon-R and
EP3 come from? The behavioral and physiological analysis in the present
study suggests that the residual trehalose sensitivity is one-fifth to one-tenth of the
Tre+ trehalose sensitivity. Since mutations in TRE does
not severely affect the sensitivity to sucrose and other sugars, other sugar receptor(s)
must also be co-expressed in the neurons. The co-expressed receptor(s) may be mainly
tuned to different subset of sugars but also weakly tuned to trehalose.
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References |
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TopIntroductionResultsDiscussionReferences |
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Clyne, P.J., Warr, C.G. and Carlson, J.R. (2000) Candidate taste receptors in Drosophila. Science, 287, 1830–1834.
Dahanukar, A., Foster, K., van der Goes van Naters, W.M. and Carlson, J.R. (2001) A Gr receptor is required for response to the sugar trehalose in taste neurons of Drosophila. Nat. Neurosci., 12, 1182–1186.
Fujishiro, N., Kijima, H. and Morita, H. (1984) Impulse frequency and action-potential amplitude in labellar chemosensory neurons of Drosophila melanogaster. J. Insect Physiol., 30, 317–325.[CrossRef]
Inomata, N., Goto, H., Itoh, M. and Isono, K. (2004) Single amino acid change of the gustatory receptor gene, Gr5a, has major effect on the trehalose sensitivity in a natural population of Drosophila melanogaster. Genetics, 167, 000–000.
Ishimoto, H., Matsumoto, A. and Tanimura, T. (2000) Molecular identification of a taste receptor gene for trehalose in Drosophila. Science, 289, 116–119.
Isono, K., Ueno, K., Morita, H. and Ohta, M. (1998) Changes in the gustatory sugar sensitivity induced by P-element excision mutations of the gene Tre in Drosophila. Jpn. J. Taste Smell Res., 5, 537–540.
Tanimura, T., Isono, K., Takamura, T. and Shimada, I. (1982) Genetic dimorphism in the taste sensitivity to trehalose in Drosophila melanogaster. J. Comp. Physiol. A, 147, 433–437.[CrossRef]
Ueno, K., Ohta, M., Morita, H., Mikuni, Y., Nakajima, S., Yamamoto, K. and Isono, K. (2001) Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a. Curr. Biol., 11, 1451–1455.[CrossRef][Web of Science][Medline]
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