Hermann Pusch

Two cDNAs coding for histamine-gated ion channels in D. melanogaster.

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 predicted from the Drosophila genome project, we identified two cDNAs (DM-HisCl-α1 and -α2) coding for putative histamine-gated chloride channels by functional expression in Xenopus laevis oocytes. DM-HisCl-α1 mRNA localizes in the lamina region of the Drosophila eye, supporting the idea that DM-HisCl-α1 may be a neurotransmitter receptor for histamine in the visual system.

Drosophila melanogaster GRD and LCCH3 subunits form heteromultimeric GABA‐gated cation channels

In addition to its action as a fast inhibitory neurotransmitter, γ‐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 ligand‐gated 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 (EC50=4.5 μM), muscimol (EC50=4.8 μM) and trans‐4‐aminocrotonic acid (EC50=104.5 μM), and partially by cis‐4‐aminocrotonic acid (EC50=106.3 μM). Picrotoxin effectively blocked the GABA‐gated channel (IC50=0.25 μM), 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.

Histamine Action on Vertebrate GABAA Receptors DIRECT CHANNEL GATING AND POTENTIATION OF GABA RESPONSES

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 β subunits of GABAA receptors as potential candidates for histamine-gated channels. In Xenopus oocytes expressing homomultimeric β channels, histamine evoked currents with an EC50 of 212 μm (β2) and 174 μm (β3), 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 β channels a unique pharmacological profile distinct from either of these receptors. In heteromultimeric channels composed of α1β2 or α1β2γ2 subunits, we found that histamine is a modulator of the GABA response rather than an agonist as it potentiates GABA-evoked currents in a γ2 subunit-controlled manner. Despite the vast number of synthetic modulators of GABAA 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 GABAA receptors and may thus represent an endogenous ligand for an allosteric site.

A Novel Chloride Channel in Drosophila melanogaster Is Inhibited by Protons

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.

The dependence of sodium pump current on internal Na concentration and membrane potential in cardioballs from sheep Purkinje fibres

The effect of various intracellular Na concentrations (cNai) 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 ofIP oncNai was investigated at a membrane potential of −40 mV by means of whote-cell recording from cardioballs internally perfused with media containing various Na concentrations. Internal perfusion with a Na free solution abolishedIP. The amplitude ofIP as a function ofcNai displayed saturation kinetics. Half maximal activation ofIP occured at acNai of about 9 mM. The maximalIP density was estimated to be 1.1μA/cm2. The potential dependence ofIP was studied by conventional whole-cell recording under various ionic conditions. GenerallyIP 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 ofIP was more pronounced at lowercNai 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 ofIP in the potential range studied. Other factors probably affecting the pump current-voltage (IP-V) relation are discussed. The results suggest thatIP varies during electrical activity.

An identification of the K activated Na pump current in sheep Prkinje fibres

The cell membrane of sheep Purkinje fibres hyperpolarizes transiently on returning to K containing media after several minutes in K free solution. To analyse this ‘K activated response’ voltage clamp experiments and measurements of the internal Na activity (aNai) are performed in fibres bathed in solution containing 0.5–2 mM BaCl2. Compared to the response in Tyrode solution the transient hyperpolarization beyond the resting potential is increased in Ba containing media. The response is blocked by 10−4 M dihydroouabain. During the response, in Ba treated fibres, aNai and a transient outward current decline with the same time constant at all clamp potentials tested. The transient outward current is most probably due to a temporary increase in electrogenic Na pumping. Both the amplitude and the time constant of the pump current show little voltage dependence. The electrogenic fraction of the active Na efflux is estimated to be about 39% and is independent of aNai. Ba ions facilitate the analysis of the pump current in voltage clamped fibres because K depletion is reduced and changes of the I–V relationship by K depletion are minimized. It is concluded that activation of the electrogenic Na pump is mainly responsible for the K activated response of fibres in Ba containing media.

Effects of Na and K Ions on the Active Na Transport in Guinea-Pig Auricles

Summary1.The effect of Na and K ions on active Na transport was studied in guinea-pig auricles by means of flame photometry.2.The Na influx into preparations rewarmed in Tyrodes solution after cooling was estimated to be about 1.05 mmole/l fibre water·min ((l.f.w.·min) or c. 8 pmole/cm2·s. Intracellular Na ions enhanced the active Na efflux over a wide range of concentrations. A decrease in the extracellular Na concentration ([Na]o) had no major effect on the active Na efflux.3.Extracellular K ions initiated an active Na efflux from rewarmed auricles with an elevated [Na]i over a narrow range of K concentrations ([K]o).4.Assuming Michaelis-Menten kinetics the maximal active Na efflux activated by internal Na ions was calculated to be about 4 mmole/l.f.w.·min (30 pmole/cm2·s). Half maximal Na efflux occurred at about 22 mmole/l.f.w. [Na]i. The maximal K-activated active Na efflux was deduced to be about 3.7 mmole/l.f.w.·min (28 pmole/cm2·s) and was half maximal at a [K]o of about 0.2 mM.5.It is tentatively concluded that the maximal active Na efflux from guinea-pig atria is 3–4 times larger than the physiological flux. Under normal conditions active Na efflux in heart is mainly regulated by variations of [Na]i.

On the temperature dependence of the Na pump in sheep Purkinje fibres

The temperature dependence of cardiac active Na transport is studied in voltage clamped sheep Purkinje fibres by means of simultaneous measurements of the membrane current (I) and the intracellular Na activity (aNai). During activation of the Na pump a transient outward current (ΔI) andaNai decline exponentially with an identical time constant (τ). The transient outward current and the decline inaNai are blocked by 10−4 M dihydroouabain (DHO). Lowering the temperature from 42°C to 17°C prolongs τ. The electrogenic fraction (e.f.) of the active Na efflux remains unaffected. The Q10 value of the active Na transport derived from the changes of τ varies within the temperature range studied. The Q10 amounts to ∼1.2 between 42°C and 35°C, to ∼2.4 between 35°C and 22°C and to ∼2.1 between 35°C and 17°C. Correspondingly the activation energy of the active Na transport is not constant between 42°C and 17°C. It is calculated to be 3.4 kcal/mol between 42°C and 35°C, 15.9 kcal/mol between 35°C and 22°C and 12.4 kcal/mol between 35°C and 17°C. Variations in temperature change the maximal rate constant of the active Na transport, whereas the sensitivity of the Na pump towards the extracellular K concentration (Ko) is little affected. The unidirectional active Na efflux of a fibre as a function of the intracellular Na concentration (Nai) at 35°C and 22°C is derived from the experemental data. The relationship is linear over the narrow Nai range studied but seems to be more complex when a wider Nai range is considered.

Unusual functional properties of homo- and heteromultimeric histamine-gated chloride channels of Drosophila melanogaster: spontaneous currents and dual gating by GABA and histamine

Histamine acts as a neurotransmitter of photoreceptors in insects and other arthropods, where it directly activates a chloride channel and mediates rapid inhibitory responses. Homo- and heteromultimeric histamine-gated ion channels formed by HisCl-alpha2 or HisCl-alpha1 + alpha2 subunits from Drosophila melanogaster were characterized by two-electrode voltage-clamp measurements of functionally expressed ion channels in Xenopus laevis oocytes. The sensitivity of heteromultimeric histamine receptors with an EC(50) of 2.3 microM is lower than that of either homomultimeric receptor. They can be further distinguished from the homomultimeric channels by their reduced sensitivity to d-tubocurarine. Heteromultimeric channels generate a spontaneous current in the absence of any agonist. This spontaneous current can be blocked in the absence of an agonist by d-tubocurarine and the histamine antagonists cimetidine, thioperamide and pyrilamine. Homomultimeric HisCl-alpha2 channels are dually gated by histamine (IC(50)=9.4 microM) and GABA (IC(50)=1.0mM), both of which are full agonists. The action of both agonists can be blocked with comparable IC(50) values by the histamine antagonists cimetidine, thioperamide and pyrilamine but not by the GABA antagonist bicuculline. Picrotoxin blocked with an IC(50) of 403 microM. Our data show that histamine and GABA act on the same ion channel, which thus might function as a site of integration of the action of different neurotransmitters.

Activation of active Na transport in sheep Purkinje fibres by external K or Rb ions.

The intracellular Na activity (aNai) of sheep Purkinje fibres bathed in solutions with and without K (Rb) is studied by means of Na sensitive microelectrodes. Perfusion with K (Rb) free media increasesaNai. Upon reapplication of K (Rb) containing solutionsaNai decreases and the cell membrane hyperpolarizes transiently.After an initial delay attributed to K (Rb) equilibration in the extracellular space, the decline ofaNai and membrane potential towards their respective resting values is approximately monoexponential and displays the same time constant (τ). The τ values andaNai in the steady state vary with the extracellular K (Rb) concentration ([K]0, [Rb]0). According to a simple model the activation of the Na pump by external K (Rb) can be estimated from the time constants or theaNai steady state values at various [K]0 ([Rb]0). Half maximal activation occurs at 1.6–3.7 mM K (Rb). External K and Rb ions are equipotent activators of the Na pump in sheep Purkinje fibres.