Histamine, a neurotransmitter and neuroregulatory compound in diverse species, serves as the neurotransmitter of photoreceptors in insects and other arthropods by directly activating a chloride channel. By systematic expression screening of novel putative ligand-gated anion channels, we identified two cDNAs (DM-HisCl-alpha 1 and-alpha 2) coding for putative histamine-gated chloride channels by functional expression in Xenopus laevis oocytes. DM-HisCl-alpha 1 mRNA localizes in the lamina region of the Drosophila eye, supporting the idea that DM-HisCl-alpha 1 may be a neurotransmitter receptor for histamine in the visual system.

In addition to its action as a fast inhibitory neurotransmitter, g-aminobutyric acid (GABA) is thought to mediate excitatory action by activating cation currents in some cell types in invertebrates. However, to date no GABA receptor capable of mediating such action has been identified at the molecular level in insects. Using a systematic expression screening approach, we found that the Drosophila ligandgated ion channel subunits GRD and LCCH3 combine to form cation-selective GABA-gated ion channels when coexpressed in Xenopus laevis oocytes. The heteromultimeric receptor is activated by GABA (EC 50 ¼ 4.5 mM), muscimol (EC 50 ¼ 4.8 mM) and trans-4-aminocrotonic acid (EC 50 ¼ 104.5 mM), and partially by cis-4-aminocrotonic acid (EC 50 ¼ 106.3 mM). Picrotoxin effectively blocked the GABA-gated channel (IC 50 ¼ 0.25 mM), but bicuculline, TPMTA, dieldrin and lindane did not. The benzodiazepines flunitrazepam and diazepam did not potentiate the GABA-evoked current. Our data suggest that heteromultimeric channels composed of GRD and LCCH3 subunits form GABA-gated cation channels in insects.

A systematic analysis of the Drosophila genome data reveals the existence of pHCl, a novel member of ligand-gated ion channel subunits. pHCl shows nearly identical similarity to glutamate-, glycine-, and histamine-gated ion channels, does however not belong to any of these ion channel types. We identified three different sites, where splicing generates multiple transcripts of the pHCl mRNA. The pHCl is expressed in Drosophila embryo, larvae, pupae, and the adult fly. In embryos, in situ hybridization detected pHCl in the neural cord and the hindgut. Functional expression of the three different splice variants of pHCl in oocytes of Xenopus laevis and Sf9 cells induces a chloride current with a linear current-voltage relationship that is inhibited by extracellular protons and activated by avermectins in a pH-dependent manner. Further, currents through pHCl channels were induced by a raise in temperature. Our data give genetic and electrophysiological evidence that pHCl is a member of a new branch of ligand-gated ion channels in invertebrates with, however, a hitherto unique combination of pharmacological and biophysical properties.Ligand-gated ion channels (LGICs) 1 mediate the fast inhibitory and excitatory responses of neuronal and muscle cells to neurotransmitters. A universal feature of the type of "Cys-loop" class of LGIC is a common topology of four membrane-spanning segments (M1-M4) and a huge N-terminal extracellular domain with a hyperconservated cysteinebridge motive (1). In vertebrates this "Cys-bridge" family of phylogenetically related genes codes for cation channels activated by acetylcholine and serotonin or for anion channels activated by GABA and glycine (1). In addition, glutamateand serotonin-gated anion channel genes are known in invertebrates (2, 3). Recently, genes for histamine-gated chloride channels and GABA-gated cation channels were identified in invertebrates (4 -7). The molecular basis of further channel types like acetylcholine-gated chloride channels in invertebrates is, however, still unknown (8). Information from the Drosophila melanogaster genome sequencing project allows identifying all members of the superfamily of ligand-gated ion channels occurring in this species by bioinformatic analysis of new homologous genes. The summarized data obtained from several published bioinformatic analyses (5,6,9,10) show that the group of ligand-gated "chloride" channels consists of 12 genes that are coding for GABA, histamine, and glutamate receptors or new, homologous ion channel types. Four members of this group cannot be directly assigned to the GABA, glutamate, or histamine branches and thus code for putative new types of ligand-gated chloride channels with yet unknown function. In a systematic expression approach of these predicted novel types of ion channels in Xenopus oocytes, it was found that none of the typical neurotransmitters activated these novel types of channels (6). Therefore, we extended the molecular biological analysis of the mRNA and found that the gene CG6112 encodes fo...

Histamine is not only a crucial cytokine in the periphery but also an important neurotransmitter and neuromodulator in the brain. It is known to act on metabotropic H1-H4 receptors, but the existence of directly histamine-gated chloride channels in mammals has been suspected for many years. However, the molecular basis of such mammalian channels remained elusive, whereas in invertebrates, genes for histamine-gated channels have been already identified. In this report, we demonstrated that histamine can directly open vertebrate ion channels and identified ␤ subunits of GABA A receptors as potential candidates for histamine-gated channels. In Xenopus oocytes expressing homomultimeric ␤ channels, histamine evoked currents with an EC 50 of 212 M (␤ 2 ) and 174 M (␤ 3 ), whereas GABA is only a very weak partial agonist. We tested several known agonists and antagonists for the histamine-binding site of H1-H4 receptors and described for ␤ channels a unique pharmacological profile distinct from either of these receptors. In heteromultimeric channels composed of ␣ 1 ␤ 2 or ␣ 1 ␤ 2 ␥ 2 subunits, we found that histamine is a modulator of the GABA response rather than an agonist as it potentiates GABA-evoked currents in a ␥ 2 subunit-controlled manner. Despite the vast number of synthetic modulators of GABA A receptors widely used in medicine, which act on several distinct sites, only a few endogenous modulators have yet been identified. We show here for the first time that histamine modulates heteromultimeric GABA A receptors and may thus represent an endogenous ligand for an allosteric site.Ligand-gated ion channels mediate the fast responses of cells to neurotransmitters (1). A universal feature of ligand-gated ion channels subunits is a common topology, comprising four membrane-spanning segments (M1-M4) and a huge N-terminal extracellular domain with a hyperconserved cysteine loop motif. In vertebrates, this "Cys loop" family of phylogenetically related genes codes for anion and cation channels activated by acetylcholine and serotonin (cation channels) or GABA 3 and glycine (anion channels) (1, 2). Despite the many years of intensive research on such ion channels, recent reports revealed unexpected new findings about this channel family. In vertebrates, a gene for zinc-gated ion channels was recently discovered (3). In insects, new classes of ligand-gated chloride channels gated by histamine or pH and cation channels gated by GABA were reported (4 -7

The effect of various intracellular Na concentrations (CiNa) and membrane potentials on the Na pump current (Ip) was studied in isolated, cultured sheep cardiac Purkinje cells ('cardioballs'). Ip was identified as cardiac steroid sensitive current. The dependence of Ip on CiNa was investigated at a membrane potential of -40 mV by means of whole-cell recording from cardioballs internally perfused with media containing various Na concentrations. Internal perfusion with a Na free solution abolished Ip. The amplitude of Ip as a function of CiNa displayed saturation kinetics. Half maximal activation of Ip occurred at a CiNa of about 9 mM. The maximal Ip density was estimated to be 1.1 microA/cm2. The potential dependence of Ip was studied by conventional whole-cell recording under various ionic conditions. Generally Ip displayed little voltage dependence at membrane potentials positive to -20 mV. Ip declined at more negative potentials. The pump cycle probably includes only one voltage sensitive step. The potential dependence of Ip was more pronounced at lower CiNa or lower concentrations of the external pump activator Cs+. The findings are in line with the idea that increasingly steeper ionic gradients against which the cations are pumped strengthen the voltage dependence of Ip in the potential range studied. Other factors probably affecting the pump current-voltage (Ip-V) relation are discussed. The results suggest that Ip varies during electrical activity.