Ligand-gated ion channels (LGICs) mediate rapid responses of neuronal and muscle cells to neurotransmitters. The large ‘cys-bridge’ superfamily of LGICs shares conserved molecular architectural features such as four membrane-spanning domains and a cys-bridge located in the large extracellular N-terminal domain³. One subgroup is formed by cation channels activated by acetylcholine and serotonin, and another, by anion channels activated by γ-aminobutyric acid (GABA)⁴, glycine³, glutamate⁵ or serotonin6. The molecular basis of histamine-gated channels, and therefore how they fit into this scheme, is still unknown. Information from the D. melanogaster genome sequencing project enables identification of all members of the superfamily of ligand-gated ion channels used by this species⁷ through bioinformatic analysis of new homologous genes. A tree constructed from the deduced protein sequences and a classification of conserved protein regions reveals that 12 genes code for GABA, glutamate, and structurally related putative new types of ligand-gated chloride channels (Supplementary Fig. 1a).

The sequences of the putative novel mRNAs and ion channel proteins were deduced from gene sequence and postulated transcript and expressed sequence tag (EST) data. The open reading frame (ORF) of the postulated transcripts was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). cDNAs were cloned into the oocyte expression vector pSGEM (transcript/protein, CT21430/AAF45992, CT5896/AAF47144, CT22815/AAF55691, CT34515/AAF54699, CT23187/AAF49337) or the pSMyc-vector⁸ (CT19189/AAF49571).

To investigate the function of the cloned cDNAs, we transcribed cRNAs in vitro and microinjected them into the cytoplasm of Xenopus oocytes. Membrane currents of oocytes were recorded by means of a two-electrode voltage clamp⁸. One-millimolar histamine
evoked current in oocytes injected with cRNA derived from CT22815/AAF55691 and CT34515/AAF54699, so the corresponding proteins were named ‘DM-HisCl-α1’ and ‘DM-HisCl- α2,’ respectively. All other cRNAs injected individually or as a pool failed to produce detectable currents in response to histamine.

Four overlapping cDNA clones isolated from a Drosophila head cDNA library fit the sequence DM-HisCl-α1, proving it correct. The sequence of DM-HisCl-α2 matched the Drosophila EST cDNA clone GH14445 except for an extra exon found in GH14445 disrupting the ORF. The deduced DM-HisCl-α1 (585 amino acids) and -α2 (427 amino acids) proteins share 51% identical amino acids and are related to Drosophila glutamate- and GABA-gated chloride channels (Supplementary Fig. 1b and c, available on the Nature Neuroscience web site).

DM-HisCl-α1 forms functional, homomeric histamine receptors when expressed in oocytes (Fig. 1a), with a median effective concentration (EC50) for histamine of 166 ± 12 μM (Fig. 1b) and a Hill coefficient of 1.9 ± 0.3. Prolonged application of histamine resulted in a weak desensitization. Oocytes did not respond to specific agonists for the vertebrate metabotropic histamine receptor types H1 and H3 (dimaprit and R-α-methylhistamine9) or to several neurotransmitters (glycine, serotonin, glutamate, GABA, dopamine, acetylcholine, adrenaline, noradrenaline or ATP) even at 1-mM concentrations. The antagonists d-tubocurarine (dTC), picrotoxin, cimetidine and pyrilamine have been used to characterize native histamine-gated ion channels^10–12. In the presence of 150 μM histamine, dTC reversibly blocked the current of DM-HisCl-α1 with a median inhibitory concentration (IC₅₀) of 3.5 ± 0.4 μM, as did cimetidine with IC₅₀ of 117 ± 14 μM and pyrilamine with IC₅₀ of 165 ± 10 μM (Fig. 1c and d). Picrotoxin partially blocked at 3 mM. Dimaprit and R-α-methylhistamine antagonized DM-HisCl-α1 with IC50 of 279 ± 12 μM and 220 ± 18 μM, respectively (Fig. 1d).

Homomeric histamine receptors formed by DM-HisCl-α2 were more sensitive to histamine, with EC₅₀ of 10.8 ± 0.46 μM (Fig. 1a and b) and Hill coefficient of 1.7 ± 0.2. The antagonists dTC, cimetidine and pyrilamine reversibly blocked the action of 10 μM histamine (IC₅₀ for dTC, 5.1 ± 0.4 μM; cimetidine, 21 ± 2.9 μM; pyrilamine, 442 ± 52 μM; Fig. 1c and f). Picrotoxin blocked at 3 mM. Dimaprit (1 mM) failed to activate DMHisCl- α2 but acted as antagonist (IC₅₀ for dimaprit, 56 ± 9.6 μM). R-α-methylhistamine acted as a partial agonist (EC₅₀, 202 ± 21 μM), activating 26% of the maximum current (Fig. 1b).

Like native histamine-gated ion channels, both DM-HisCl-α1 and -α2 are chloride-selective ion channels. The histamine-dependent current reverses near –20 mV (Fig. 1e), close to the predicted chloride reversal potential for Xenopus oocytes in 120 mM extracellular chloride. Variation of the chloride concentration shifted the reversal potential of the DM-HisCl-α1 channel in good agreement with the theoretical shift predicted by the Nernst equation for chloride channels (Fig. 1g; see also Supplementary Fig. 1 legend).

RT-PCR analysis detected the expression of both channel transcripts in the adult head and body and late pupal stage, but not in egg or larva (Fig. 2a). ISH (in situ hybridization) to head cryosections showed that DM-HisCl-α1 is expressed in the first optic neuropil (lamina) region of the Drosophila visual system. Weak signals could also be detected throughout the neuropil (Fig. 2b and d; see also Supplementary Fig. 3 legend). Despite exhaustive efforts, DM-HisCl-α2 could not be detected in the brain by ISH, although its mRNA was amplified in head by RT-PCR.

Histamine is implicated as the major neurotransmitter in the eyes of several arthropods, including Drosophila, blowfly, moth, cockroach, locust, barnacle and Limulus^2,13. ISH detects DMHisCl- α1 mRNA in the lamina region of the optic lobes, coinciding with the distribution of histamine established by immunocytochemical detection¹⁴. This suggests that the receptor is active in visual signal transduction at the first postsynaptic cells, the large monopolar cells (LMCs). The recombinant DM-HisCl-α1 has a higher EC₅₀ (166 μM versus 24 μM) than was measured for Drosophila LMCs¹⁵. One reason may be that EC50 values determined in various expression systems differ. In addition, the discrepancy may reflect a different subunit composition of the native channel. The sensitivity to histamine of DM-HisCl-α2 (EC₅₀, 11 μM versus 24 μM) is closer to that of the native LMC receptor, but it is unclear whether DM-HisCl-α2 is expressed in the lamina.

Recombinant and native channels have a similar pharmacological profile. The antagonists cimetidine, pyrilamine and dTC block both the recombinant channels and the light- or histamineinduced responses in the LMC of the housefly in vivo¹⁰. The histamine- gated channel of the lobster cardiac ganglion¹² is similar to HisCl-α2 with respect to the sensitivity for histamine, cimetidine and dTC. In summary, the electrophysiological and pharmacological properties of DM-HisCl-α1/-α2 imply that they are prototypes of a new class of inhibitory ligand-gated ion channels, not only in insects but also in other arthropods such as the lobster.

The GenBank accession number for DM-HisCl-α1 is AF435469; for DM-HisCl-α2, AF435470; for the GH14445 type slice variant, AF435471.

1. Schwartz, J. C., Arrang, J. M., Garbarg, M., Pollard, H. & Ruat, M. Physiol. Rev.
   71, 1–51 (1991).

2. Hardie, R. C. Nature 339, 704–706 (1989).

3. Ortells, M. O. Trends Neurosci. 18, 121–127 (1995).

4. Ffrench-Constant, R. H., Mortlock, D. P., Shaffer, C. D., MacIntyre, R. J. &
   Roush, R. T. Proc. Natl. Acad. Sci. USA 88, 7209–7213 (1991).

5. Cully, D. F., Paress, P. S., Liu, K. K., Schaeffer, J. M. & Arena, J. P. J. Biol. Chem.
   271, 20187–20191 (1996).

6. Ranganathan, R., Cannon, S. C. & Horvitz, H. R. Nature 408, 470–475 (2000).

7. Littleton, J. T. & Ganetzky, B. Neuron 26, 35–43 (2000).

8. Wetzel, C. H., et al. J. Neurosci. 19, 7426–7433 (1999).

9. van der Goot, H. & Timmerman, H. Eur. J. Med. Chem. 35, 5–20 (2000).

10. Hardie, R. C. J. Exp. Biol. 138, 221–241 (1988).

11. Ache, B. W. & McClintock, T. S. Proc. Natl. Acad. Sci. USA 86, 8137–8141 (1989).

12. Hashemzadeh-Gargari, H. & Freschi, J. E. J. Neurophysiol. 68, 9–15 (1992).

13. Stuart, A. E. Neuron 22, 431–433 (1999).

14. Melzig, J., Burg, M., Gruhn, M., Pak, W. L. & Buchner, E. J. Neurosci. 18,
    7160–7166 (1998).

15. Skingsley, D. R., Laughlin, S. B. & Hardie, R. C. J. Comp. Physiol. A 176, 611–623
    (1995).

Fig. 1. Electrophysiological and pharmacological characterization of cloned DM-HisCl-α1/-α2 expressed in Xenopus oocytes as described8. (a) Agonist profile. Histamine was applied to voltage-clamped (–80 mV) oocytes injected with in vitro transcripts from DM-HisCl-α1 and DM-HisCl-α2. (b) Concentration–response curves for histamine and R-α-methylhistamine. DM-HisCl-α1, triangle; DM-HisCl-α2, square; DM-HisCl-α2, circle. (c) Action of cimetidine. Oocytes were voltage-clamped (–80 mV) and histamine was applied. After washout, histamine was applied in combination with the antagonist. (d) Concentration–response curves of DM-HisCl-α1 for antagonists. Cimetidine, circle; pyrilamine, square; dTC, triangle; R-α-methylhistamine, diamond; dimaprit, inverted triangle. (e) The averaged peak currents of I–V curves for HisCl-α1 (square) and HisCl-α2 (triangle) show a linear I–V relationship. (f) Concentration-response curve of DM-HisCl-α2 for antagonists. Cimetidine, circle; pyrilamine, square; dTC, triangle; dimaprit, inverted triangle. (g) Chloride-dependent reversal potentials of HisCl-α1. Sodium chloride was partially replaced by sodium gluconate.

Fig. 2. RT-PCR and in situ hybridization. (a) Expression of DM-HisCl-α1/-α2 transcripts was investigated by RT-PCR in various developmental stages and in head and body of adult flies. The ribosomal protein 49 (U92431) was used as a control for cDNA quality. (b, c) Overview of a horizontal cross section through the head with hybridization of the digoxigenin-UTP labeled HisCl-α1 antisense (b) and sense probe (c). (d, e) Enlarged views of head cross sections. Lateral cells of the lamina are intensely stained (arrowheads) and cells in the total neuropil are weakly stained (b, d). (e) Additional staining of dorsal neurosecretory cells (arrowheads). ret, retina; me, medulla; la, lamina. Scale bars, 200 μm (b, c), 60 μm (d), 100 μm (e).