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Comparative Study
, 58 (6), 884-96

DAG Lipase Activity Is Necessary for TRP Channel Regulation in Drosophila Photoreceptors

Affiliations
Comparative Study

DAG Lipase Activity Is Necessary for TRP Channel Regulation in Drosophila Photoreceptors

Hung-Tat Leung et al. Neuron.

Abstract

In Drosophila, a phospholipase C-mediated signaling cascade links photoexcitation of rhodopsin to the opening of the TRP/TRPL channels. A lipid product of the cascade, diacylglycerol (DAG) and its metabolite(s), polyunsaturated fatty acids (PUFAs), have both been proposed as potential excitatory messengers. A crucial enzyme in the understanding of this process is likely to be DAG lipase (DAGL). However, DAGLs that might fulfill this role have not been previously identified in any organism. In this work, the Drosophila DAGL gene, inaE, has been identified from mutants that are defective in photoreceptor responses to light. The inaE-encoded protein isoforms show high sequence similarity to known mammalian DAG lipases, exhibit DAG lipase activity in vitro, and are highly expressed in photoreceptors. Analyses of norpA inaE double mutants and severe inaE mutants show that normal DAGL activity is required for the generation of physiologically meaningful photoreceptor responses.

Figures

Fig. 1
Fig. 1. ERG analyses of inaEN125
A) Comparison of ERG phenotypes of inaEN125 (N125), trpP343 (P343), and wild type (wt). ERGs recorded from wild type, inaEN125, and trpP343 are shown superimposed. The inaEN125 phenotype fell between those of wild type and trp in severity. B, Or: blue and orange stimuli generated by interposing, respectively, Corning 5433 and CS2–73 filters in the light path. B) Comparison of refractory periods among wild type, inaEN125, and trpP343. For each genotype, the fly was given a 5 s orange stimulus and allowed to dark adapt for 3 min before being exposed to the first stimulus. Immediately after the first stimulus, a 5 s orange stimulus was given and the second stimulus was delivered after a dark duration of 10, 30, 70, 110, 150, or 180 s. C-a) Semidominance of TrpP365 (P365). P365 heterozygotes (P365/+) generated responses that were about half-way between those of wild type and P365 homozygotes in size. C-b) Enhancement of P365/+ by N125. C-c) Non-complementation between KG08585 (P-line) and N125. KG08585 enhanced the P365/+ phenotype. Moreover, a heteroallelic combination of KG08585 and N125 also enhanced P365/+. C-d,e) The interaction between inaEN125 and TrpP365 was nullified by the expression of the D form (d) but not the A form of INAE. (e). D) Rescue of the inaEN125 ERG phenotype by the rescue construct harboring the inaE-RD isoform but not by that containing the inaE-RA isoform. Quantification of the above data based on multiple recordings is presented in Table 1.
Fig. 2
Fig. 2. Maps of the inaE region, gene, transcript, and protein
A) The inaE genomic region. inaE mutations are uncovered by the deficiencies Df(1)benCO2 (first line) and Df(1)AR10(second line). The third line shows cytogenetic regions, and the fourth line indicates nucleotide coordinates. B) The CG33174 (inaE) gene and its transcripts. Yp3 and CG32626, shown in black, were identified along with CG33174 in microarrays as potential candidate genes. The inaE gene generates two transcripts, RA and RD, by alternative splicing. In the A form, the 13th intron is not spliced out and transcription ends in the 13th intron, thereby adding one amino acid residue to the A form of the protein (indicated by “643 + 1” in (C)). Untranslated regions are shown in grey. The sites of the inaEN125 mutation and of KG08585 insertion are indicated. C) The INAE protein. The regions of the four transmembrane segments and the lipase domain, as well as the 20-mer peptide used in antibody generation, are indicated. The C-termini of the A and D forms of the INAE protein are indicated by arrows and the C-terminal amino acid residue number. The vertical lines correspond to coding exon boundaries.
Fig. 3
Fig. 3. DAG lipase assay
A) Potential metabolic pathways of the DAG substrate. B) Enzymatic characterization of INAE isoforms. Representative LC-MS total ion chromatograms (m/z 65–500) of INAE-D (B-a) and INAE-A (B-b) lipase assay products, using 1-stearoyl-2-arachidonoyl-sn-glycerol as substrate. Arachidonic acid (20:4), stearic acid (18:0), 1-stearoyl glycerol (1-SG), and 2-arachidonoyl glycerol (2-AG) were eluted at 5.8, 6.3, 8.7, and 9.3 min, respectively. Samples contained palmitic acid as an internal standard (i.s.). C) Velocity dependence plots as a function of substrate concentration obtained by GC-MS. Assays were performed on recombinant INAE-D (C-a) and INAE-A (C-b) using the same substrate as in (B). Hydrolysis rates at sn-1 and sn-2 positions were estimated by the amount of released stearic acid and arachidonic acid, respectively (Fig. 3A). Open and closed circles represent sn-1 and sn-2 reactions, respectively. Values represent the mean ± SD (n = 4). Note that the ordinate scales are different between (B-a) and (B-b) and between (C-a) and (C-b). C-c) Determination of kinetic parameters by Lineweaver-Burk plots. Calculations were performed by pooling sn-1 and sn-2 data for each isoform. V: velocity; [S]: substrate concentration.
Fig. 4
Fig. 4. Western blot analysis of inaE mutants with eya and wt controls
A) A representative blot obtained in Western blot analyses carried out on the isolated heads of the inaE mutants, N125, P19, and KG08585. Beta actin was used as loading control; eya: eyes absent. B) Quantification of the data. Each data point was normalized with respect to the corresponding beta actin loading control, and the normalized data points in a given blot were in turn normalized to the wild-type PD value. Based on three repetitions.
Fig. 5
Fig. 5. Fluorescence confocal microscopy of the photoreceptor layer
A) A cross-sectional image taken ~35 μm from the distal tip of the ommatidia. Insets show the two highlighted ommatidia in higher magnification. Occasionally, some puncta are seen well inside the rhabdomres (arrow). B) A longitudinal section showing nearly the entire length of the ommatidia. Green: anti-INAE; red: F-actin in rhabodomeres. Rh: rhabdomere.
Fig. 6
Fig. 6. Electron microscopy of xl ommatidia
A) Transverse sections of groups of ommatidia obtained at ~35 μm depth from the distal tips of rhabdomeres in 2–3 d post-eclosion wild type, xl129, xl15 and xl18. B) Fractions of ommatidia containing 7, 5–6, and 1–4 rhabdomeres in wild type, xl29, xl15 and xl18. The number of rhabdomeres in each ommatidium was counted in low magnification views of the entire section in 4, 4, 3, and 4 different eyes of wild type, xl29, xl15, and xl18, respectively.
Fig. 7
Fig. 7. A) Molecular alterations in xl mutants
xl29 had no mutation in the inaE gene, xl15 carried a >1.5 kb insertion just upstream of Exon 13 (determined within 50 bp), and xl18 carried a ~1.5 kb deletion, which was replaced by a 0.68 kb sequence translocated from the right arm of the third chromosome. B) Quantitative RT PCR measurements, based on three repetitions. All values obtained with primer pairs 1/2 and 3/4 were normalized with respect to those of inaE-D specific transcript of wild type. Those values obtained with the primer pair 5/6 were normalized with respect to wild type values within that series. No inaE-A transcript was detected in xl18 because the A-form specific sequence was deleted in the mutant. Because the primer pair 5/6 amplified a much larger product than 1/2 or 3/4 (~400 bp vs. <150 bp), the amplification was less efficient. Locations of primer pairs 1/2 and 3/4 are shown in (A). The primer pairs 5/6 were located in the 4th and 7th exons, respectively.
Fig. 8
Fig. 8. Analyses of inaE mutants and double mutants
A) A diagram depicting a part of the phototransduction cascade and the phosphoinositide recycling pathway. Names of the genes encoding the proteins are shown in parentheses, and the mutants used in the present study are shown in italics under the protein names. H43: norpAH43; N125: inaEN125; xl18: inaExl18. B) norpA inaE double mutant study. B-i) Intracellular recordings of the receptor potentials elicited from the H43 mutant are compared with those obtained from the H43 N125 or H43 xl18 double mutant. Receptor potentials of 12.5 ± 2.9 mV peak amplitude and 6.4 ± 2.3 mV steady-state amplitude were obtained from H43 by a bright white stimulus of 10 s duration. The same stimulus elicited a fast transient response of 11.5 ± 2.4 mV peak amplitude with no steady-state component from the H43 N125 double mutant and no response at all from the H43 xl18 double mutant. B-ii) Initial portions of the responses shown in B-i are displayed in an expanded time scale. C) inaE mutations, xl15, xl18 and xl29. C-i) Intracellular recordings of receptor potentials elicited by a bright, white, 30 s stimulus from xl18, xl15, and xl29 are shown superimposed. B-ii) The initial portions of the responses in C-i are shown expanded in time scale by 40x. Quantification of the data, including those from N125, is presented in Table 2. D) Enhancement of the P365/+ phenotype. Introducing N125 to the P365/+ background abolishes the small, ~7 mV receptor potential present in P365/+. Replacing N125 with xl18 makes the phenotype of the double mutant less severe. EGUF is wild type for P365. Because it is needed in xl18;P365/EGUF to make the fly mosaic for xl18, it was also introduced in other genotypes to maintain the same genetic background.

Comment in

  • In Search of the Holy Grail for Drosophila TRP
    C Montell. Neuron 58 (6), 825-7. PMID 18579072. - Review
    Activation of the archetypal Transient Receptor Potential (TRP) channel, which is essential for Drosophila phototransduction, depends on a phospholipase C (PLC). However, …

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