The β-alanine transporter BalaT is required for visual neurotransmission in Drosophila
- Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, China
- National Institute of Biological Sciences, China
- Peking University, China
- Beijing Normal University, China
Figures
Figure 1 with 3 supplements
CG3790 is a retina-enriched β-alanine transporter.
(A) Screening for a β-alanine transporter. HEK293T cells transiently expressing one of ~20 head-enriched potential transporters were exposed to [3H]-β-alanine, which was added to the ECF buffer …
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Figure 1—source data 1
Relates to Figure 1A and B.
[3H]-β-alanine uptake data for screening for β-alanine transporter in HEK 293 T cells and S2 cells (.xlsx file).
- https://doi.org/10.7554/eLife.29146.004
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Figure 1—source data 2
Relates to Figure 1D.
Relative CG3790 transcript levels from wild-type (wt) bodies, wt heads, and GMR-hid heads. RNA levels were normalized to levels in wt bodies, which were set to 1 (.xlsx file).
- https://doi.org/10.7554/eLife.29146.005
Figure 1—figure supplement 1
CG3790 is a SLC22 family protein.
Alignment of the Drosophila CG3790 amino acid sequence with mouse OCT3 (mOCT3) and human OCT3 (hOCT3). Identical residues, found in at least two proteins, are enclosed in green boxes. CG3790 is 29% …
Figure 1—figure supplement 2
CG3790 is unable to transport histamine and GABA.
(A) CG3790 did not transport GABA into S2 cells. Mouse GAT3 was used as a positive control, and RFP was used as a negative control. (B) CG3790 did not exhibit histamine transporting activity in S2 …
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Figure 1—figure supplement 2—source data 1
Relates to Figure 1—figure supplement 2A and B.
[3H]-histamine uptake data in S2 cells transfected mock and RFP negative control or mGAT3. [3H]-GABA uptake data in S2 cells transfected mock and RFP negative control or hOCT2 (.xlsx file).
- https://doi.org/10.7554/eLife.29146.008
Figure 1—figure supplement 3
GMR-hid abolished expression of ninaE and pdh.
(A–B) Wild-type heads contain more transcripts for (A) ninaE and (B) pdh than wild-type bodies and GMR-hid heads (gpdh served as an internal control, and mRNA levels were normalized to wild-type …
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Figure 1—figure supplement 3—source data 1
Relates to Figure 1—figure supplement 3A and B.
Relative ninaE and pdh transcript levels from wild-type (wt) bodies, wt heads, and GMR-hid heads. RNA levels were normalized to levels in wt bodies. Data is processed by logarithmic 2 (.xlsx file).
- https://doi.org/10.7554/eLife.29146.010
Figure 2 with 1 supplement
Mutations in balat disrupt photoreceptor cell synaptic transmission and visual behavior.
(A) Schematic for balat knock-out by sgRNA targeting. The organization of the balat locus and the expected structures of balta1 and balat2 alleles are shown. Boxes represent exons with the coding …
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Figure 2—source data 1
Relates to Figure 2D and E.
Quantitative analysis of the amplitudes of ERG OFF transients about wild-type (w1118), balat1, balat2, balat1;GMR-balaT, balat1;pdh-balaT, balat2;ninaE-balaT and w+; balat1 flies. Quantitative of phototaxis behaviors of wt, balat1 balat1;GMR-balaT, balat1;pdh-balaT and balat2;ninaE-balaT flies (.xlsx file).
- https://doi.org/10.7554/eLife.29146.013
Figure 2—figure supplement 1
Quantification of the ERG ON transients.
Percentage of flies of indicated genotypes that have ON transients were quantified. The ERG profiles from at least 7 flies of each genotype were scored.
Figure 3 with 1 supplement
BalaT localizes to pigment cells.
(A–B) Compound eyes from balat-mcherry flies that express mCherry (red) driven by the endogenous balat promoter were labeled for PDH (green), mCherry (red), and phalloidin (blue). High-magnification …
Figure 3—figure supplement 1
Generation of balat-mcherry flies.
(A) Schematics for generating balat-mcherry flies. The mcherry fluorescent protein, driven by an eye-specific 3XP3 promoter, was inserted into the balat genomic locus using CRISPR/Cas9-mediated …
Figure 4 with 1 supplement
Specific knockdown of Inx1 and Inx3 in compound eyes blocks visual neurotransduction.
ERG recordings of (A) control (GMR-gal4), (B) inx1RNAi (GMR-gal4/ UAS-inx1RNAi), (C) inx2RNAi (GMR-gal4/UAS-inx2RNAi), (D) inx3RNAi (GMR-gal4/ UAS-inx3RNAi), (E) inx1RNAi + inx2RNAi (GMR-gal4/UAS-inx…
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Figure 4—source data 1
Relates to Figure 4J.
Quantitative of fluorescence intensity ratios of β-alanine signals between retina and lobula of inx1RNAi + inx3RNAi and controls (CG2198RNAi and CG9962RNAi) (.xlsx file).
- https://doi.org/10.7554/eLife.29146.018
Figure 4—figure supplement 1
Accumulation of β-alanine in heads of Inx1 and Inx3 double RNAi flies.
Levels of β-alanine in heads of control and inx1RNAi + inx3RNAi (GMR-gal4/UAS-inx1RNAi UAS-inx3RNAi) flies. Error bars indicate SDs, and significant differences between mutant and wt flies were …
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Figure 4—figure supplement 1—source data 1
Relates to Figure 4—figure supplement 1.
Quantitative of levels of β-alanine in heads of control and inx1RNAi + inx3RNAi (GMR-gal4/UAS-inx1RNAi UAS-inx3RNAi) flies (.xlsx file).
- https://doi.org/10.7554/eLife.29146.020
Figure 5 with 1 supplement
Loss of BalaT affects the level and distribution of β-alanine in vivo.
(A–B) Levels of (A) β-alanine and (B) histamine in heads of wild-type (wt, w1118), cart1, and balat1 flies are shown. Error bars indicate SDs, and significant differences between mutant and wt flies …
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Figure 5—source data 1
Relates to Figure 5A and B.
Quantitative of levels of β-alanine and histamine in heads of wild-type, cart1, and balat1 flies (.xlsx file).
- https://doi.org/10.7554/eLife.29146.022
Figure 5—figure supplement 1
Distribution of β-alanine was unaffected in pyd1 and balck double mutant flies.
β-alanine was immunolabeled in horizontal sections of heads from (A) wild type (w1118) and (B) pyd1;b1 flies. Re, retina; La, lamina; Me, medulla; Lo, lobula. (C) Fluorescence intensity ratios of …
Figure 6
A model of the histamine recycling pathway.
Histamine is initially synthesized by histidine decarboxylase (Hdc) in photoreceptor cells (PR). Upon light stimulation, PRs release histamine into the synaptic cleft. Released histamine is quickly …
Tables
Table 1
Description of 20 head-enriched genes.
| CG number | Name | Description |
|---|---|---|
| CG3790 | Organic anion transmembrane transporter activity | |
| CG18660 | Nckx30C | Sodium/potassium/calcium exchanger |
| CG2893 | zydeco | Potassium-dependent sodium/calcium exchanger |
| CG7342 | Organic anion transmembrane transporter activity | |
| CG13610 | Orct2 | Organic cation transporter |
| CG6331 | Orct | Organic anion transmembrane transporter activity |
| CG6126 | Organic anion transmembrane transporter activity | |
| CG4360 | Nucleic acid binding | |
| CG3159 | Eaat2 | L-aspartate transmembrane transporter activity |
| CG6723 | Sodium/solute symporter | |
| CG11010 | Ent3 | Nucleoside transmembrane transporter activity |
| CG13743 | Amino acid transporter | |
| CG10804 | Neurotransmitter transporter activity | |
| CG4545 | SerT | Monoamine transmembrane transporter activity |
| CG5281 | EamA domain | |
| CG43066 | Neurotransmitter transporter activity | |
| CG7442 | SLC22A | Choline transmembrane transporter activity |
| CG7708 | Sodium/solute symporter | |
| CG13248 | Amino acid/polyamine transporter I | |
| CG42322 | Solute carrier family 35 member F3/F4 |
