The Benzodiazepine Binding Sites of GABAA Receptors.

Everyday activity is based on a subtle equilibrium of excitatory and inhibitory neuronal systems. The most prominent players in neuronal inhibition are synaptic and extrasynaptic GABAA receptors. Benzodiazepines are popular drugs that act as positive allosteric modulators of a subset of these receptors. Benzodiazepines have sedative, hypnotic, muscle-relaxant, and anticonvulsive effects, and are of outstandingly low overdose risk. The discovery of a large number of subtypes of GABAA receptors has raised hopes for a clear separation of this spectrum of actions. We discuss here how far this separation has been achieved, and outline recent progress towards the discovery of novel ligands for canonical and non-canonical binding sites.

Transporters of Trypanosoma brucei-phylogeny, physiology, pharmacology.

Trypanosoma brucei comprise the causative agents of sleeping sickness, T. b. gambiense and T. b. rhodesiense, as well as the livestock-pathogenic T. b. brucei. The parasites are transmitted by the tsetse fly and occur exclusively in sub-Saharan Africa. T. brucei are not only lethal pathogens but have also become model organisms for molecular parasitology. We focus here on membrane transport proteins of T. brucei, their contribution to homeostasis and metabolism in the context of a parasitic lifestyle, and their pharmacological role as potential drug targets or routes of drug entry. Transporters and channels in the plasma membrane are attractive drug targets as they are accessible from the outside. Alternatively, they can be exploited to selectively deliver harmful substances into the trypanosome's interior. Both approaches require the targeted transporter to be essential: in the first case to kill the trypanosome, in the second case to prevent drug resistance due to loss of the transporter. By combining functional and phylogenetic analyses, we were mining the T. brucei predicted proteome for transporters of pharmacological significance. Here, we review recent progress in the identification of transporters of lipid precursors, amino acid permeases and ion channels in T. brucei.

Identification and characterization of the three members of the CLC family of anion transport proteins in Trypanosoma brucei.

CLC type anion transport proteins are homo-dimeric or hetero-dimeric with an integrated transport function in each subunit. We have identified and partially characterized three members of this family named TbVCL1, TbVCL2 and TbVCL3 in Trypanosoma brucei. Among the human CLC family members, the T. brucei proteins display highest similarity to CLC-6 and CLC-7. TbVCL1, but not TbVCL2 and TbVCL3 is able to complement growth of a CLC-deficient Saccharomyces cerevisiae mutant. All TbVCL-HA fusion proteins localize intracellulary in procyclic form trypanosomes. TbVCL1 localizes close to the Golgi apparatus and TbVCL2 and TbVCL3 to the endoplasmic reticulum. Upon expression in Xenopus oocytes, all three proteins induce similar outward rectifying chloride ion currents. Currents are sensitive to low concentrations of DIDS, insensitive to the pH in the range 5.4 to 8.4 and larger in nitrate than in chloride medium.

α subunits in GABAA receptors are dispensable for GABA and diazepam action.

The major isoform of the GABAA receptor is α1ß2γ2. The binding sites for the agonist GABA are located at the ß2+/α1- subunit interfaces and the modulatory site for benzodiazepines at α1+/γ2-. In the absence of α1 subunits, a receptor was formed that was gated by GABA and modulated by diazepam similarly. This indicates that alternative subunits can take over the role of the α1 subunits. Point mutations were introduced in ß2 or γ2 subunits at positions homologous to α1- benzodiazepine binding and GABA binding positions, respectively. From this mutation work we conclude that the site for GABA is located at a ß2+/ß2- subunit interface and that the diazepam site is located at the ß2+/γ2- subunit interface. Computational docking leads to a structural hypothesis attributing this non-canonical interaction to a binding mode nearly identical with the one at the α1+/γ2- interface. Thus, the ß2 subunit can take over the role of the α1 subunit for the formation of both sites, its minus side for the GABA binding site and its plus side for the diazepam binding site.

TbIRK is a signature sequence free potassium channel from Trypanosoma brucei locating to acidocalcisomes.

Potassium channels from prokaryotes and eukaryotes are usually recognized by a typical amino acid sequence TXTGY(F)G representing the ionic selectivity filter. Using a screening approach with ion channel family profiles but without the above motif, we identified a gene in Trypanosoma brucei that exhibits homology to inward rectifying potassium channels. We report here cloning of this ion channel named TbIRK. The protein is localized to acidocalcisomes in procyclic and in bloodstream form parasites. Functional properties of this channel were established after expression in Xenopus oocytes. Currents recorded in potassium medium show inward rectification and little time dependence. Surprisingly, this channel retains selectivity for potassium ions over sodium ions >7, in spite of the lack of the classical selectivity filter. The sequence GGYVG was predicted in silico to replace this filter motif. Point mutations of the corresponding glycine residues confirmed this at the functional level. The channel is inhibited by caesium ions but remains unaffected by barium ions up to 10 mM. TbIRK is to our knowledge the first potassium channel in T. brucei that localizes to the acidocalcisomes, organelles involved in the storage of phosphates and the response to osmotic stress that occurs during the life cycle of trypanosomes.

H+-dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter.

Trypanosoma brucei is an extracellular protozoan parasite that causes human African trypanosomiasis or "sleeping sickness". During the different phases of its life cycle, T. brucei depends on exogenous inorganic phosphate (Pi), but little is known about the transport of Pi in this organism. In the present study, we showed that the transport of 32Pi across the plasma membrane follows Michaelis-Menten kinetics and is modulated by pH variation, with higher activity at acidic pH. Bloodstream forms presented lower Pi transport in comparison to procyclic forms, that displayed an apparent K0.5 = 0.093 ± 0.008 mM. Additionally, FCCP (H+-ionophore), valinomycin (K+-ionophore) and SCH28080 (H+, K+-ATPase inhibitor) inhibited the Pi transport. Gene Tb11.02.3020, previously described to encode the parasite H+:myo-inositol transporter (TbHMIT), was hypothesized to be potentially involved in the H+:Pi cotransport because of its similarity with the Pho84 transporter described in S. cerevisiae and other trypanosomatids. Indeed, the RNAi mediated knockdown remarkably reduced TbHMIT gene expression, compromised cell growth and decreased Pi transport by half. In addition, Pi transport was inhibited when parasites were incubated in the presence of concentrations of myo-inositol that are above 300 µM. However, when expressed in Xenopus laevis oocytes, two-electrode voltage clamp experiments provided direct electrophysiological evidence that the protein encoded by TbHMIT is definitely a myo-inositol transporter that may be only marginally affected by the presence of Pi. These results confirmed the presence of a Pi carrier in T. brucei, similar to the H+-dependent inorganic phosphate system described in S. cerevisiae and other trypanosomatids. This transport system contributes to the acquisition of Pi and may be involved in the growth and survival of procyclic forms. In summary, this work presents the first description of a Pi transport system in T. brucei.

Low Expression in Xenopus Oocytes and Unusual Functional Properties of α1ß2γ2 GABAA Receptors with Non-Conventional Subunit Arrangement.

The major subunit isoform of GABAA receptors is α1ß2γ2. The subunits are thought to surround an ion pore with the counterclockwise arrangement α1γ2ß2α1ß2 as seen from the outside of the neuron. These receptors have two agonist sites and one high affinity drug binding site specific for benzodiazepines. Recently, this receptor was postulated to assume alternative subunit stoichiometries and arrangements resulting in only one agonist site and one or even two sites for benzodiazepines. In order to force a defined subunit arrangement we expressed a combination of triple and dual concatenated subunits. Here we report that these unconventional receptors express only small current amplitudes in Xenopus oocytes. We determined agonist properties and modulation by diazepam of two of these receptors that resulted in currents large enough for a characterization, that is, ß2-α1-γ2/α1-γ2 and ß2-α1-γ2/ß2-γ2. The first pentamer predicted to have two benzodiazepine binding sites shows similar response to diazepam as the standard receptor. As expected for both receptors with a single predicted agonist site the concentration response curves for GABA were characterized by a Hill coefficient < 1. ß2-α1-γ2/ß2-γ2 displayed a mM apparent GABA affinity for channel opening instead of the expected µM affinity. Based on their subunit and binding site stoichiometry, that contradicts all previous observations, their unusual functional properties and their very low expression levels in oocytes, we consider it unlikely that these unconventional receptors are expressed in neurons to an appreciable extent.

Toward Understanding Functional Properties and Subunit Arrangement of α4ß2δ γ-Aminobutyric Acid, Type A (GABAA) Receptors.

GABAA receptors are pentameric ligand-gated channels mediating inhibitory neurotransmission in the CNS. α4ßxδ GABAA receptors are extrasynaptic receptors important for tonic inhibition. The functional properties and subunit arrangement of these receptors are controversial. We predefined subunit arrangement by using subunit concatenation. α4, ß2, and δ subunits were concatenated to dimeric, trimeric, and, in some cases, pentameric subunits. We constructed in total nine different receptor pentamers in at least two different ways and expressed them in Xenopus oocytes. The δ subunit was substituted in any of the five positions in the α1ß2 receptor. In addition, we investigated all receptors with the 2:2:1 subunit stoichiometry for α4, ß2, and δ. Several functional receptors were obtained. Interestingly, all of these receptors had very similar EC50 values for GABA in the presence of the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC). All functional receptors containing δ subunits were sensitive to 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide (DS2). Moreover, none of the receptors was affected by ethanol up to 30 mm These properties recapitulate those of non-concatenated receptors expressed from a cRNA ratio of 1:1:5 coding for α4, ß2, and δ subunits. We conclude that the subunit arrangement of α4ß2δ GABAA receptors is not strongly predefined but is mostly satisfying the 2:2:1 subunit stoichiometry for α4, ß2, and δ subunits and that several subunit arrangements result in receptors with similar functional properties tuned to physiological conditions.

Molecular mode of action of CGS 9895 at α1 ß2 γ2 GABAA receptors.

γ-aminobutyric type A (GABAA ) receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics, and anesthetics. Previously, pyrazoloquinoline 2-p-methoxyphenylpyrazolo [4,3-c] quinolin-3(5H)-one (CGS 9895) was described as a positive allosteric modulator acting through the α+/ß- interface in the extracellular domain of GABAA receptors. The localization of the binding site was based on a steric hindrance approach, rather than on direct effects of point mutations. In this study we further characterized modulation by this compound which seems to have multiple sites of action. We investigated GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. We have identified the α1 Y209 residue at the α+/ß- interface as a key residue for CGS 9895 modulation. In addition, the interaction between this residue and various structural analogs was characterized, allowing tentative positioning of CGS 9895 versus α1 Y209 (rat sequence). Not all compounds were found to be sensitive to mutations at the α1 Y209 residue. In addition, the interaction of CGS 9895 with flurazepam was characterized. Flurazepam is hypothesized to act at the same subunit interface in the extracellular domain. We also provide evidence that the GABAA receptor harbors additional modulatory sites for CGS 9895 at each of the subunit interfaces in the transmembrane domain. GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We have identified the α1 Y209 residue present at the extracellular α+/ß- subunit interface as a key residue for the positive allosteric modulation of the GABAA receptor by CGS 9895. This receptor harbors additional modulatory sites for this compound at subunit interfaces in the transmembrane domain.

Novel positive allosteric modulators of GABAA receptors with anesthetic activity.

GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1ß2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1ß2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two ß+/α- subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual ß+/α- interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics.

Functional sites involved in modulation of the GABAA receptor channel by the intravenous anesthetics propofol, etomidate and pentobarbital.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs. Among the many modulatory compounds are also the intravenous anesthetics propofol and etomidate, and barbiturates. The mechanism of receptor modulation by these compounds is of mayor relevance. The site of action of these compounds has been located to subunit interfaces in the intra-membrane region of the receptor. In α1ß2γ2 GABAA receptors there are five such interfaces, two ß+/α- and one each of α+/ß-, α+/γ- and γ+/ß- subunit interfaces. We have used reporter mutations located in the second trans-membrane region in different subunits to probe the effects of changes at these subunit interfaces on modulation by propofol, etomidate and pentobarbital. We provide evidence for the fact that each of these compounds either modulates through a different set of subunit interfaces or through the same set of subunit interfaces to a different degree. As a GABAA receptor pentamer harbors two ß+/α- subunit interfaces, we used concatenated receptors to dissect the contribution of individual interfaces and show that only one of these interfaces is important for receptor modulation by etomidate.

A new chromanone derivative isolated from Hypericum lissophloeus (Hypericaceae) potentiates GABAA receptor currents in a subunit specific fashion.

A phytochemical investigation of the lipophilic extract of Hypericum lissophloeus (smoothbark St. John's wort, Hypericaceae) was conducted, resulting in the isolation and identification of a new chromanone derivative: 5,7-dihydroxy-2,3-dimethyl-6-(3-methyl-but-2-enyl)-chroman-4-one (1). This compound was demonstrated to act as a potent stimulator of currents elicited by GABA in recombinant α1ß2γ2 GABAA receptors, with a half-maximal potentiation observed at a concentration of about 4µM and a maximal potentiation of >4000%. Significant potentiation was already evident at a concentration as low as 0.1µM. Extent of potentiation strongly depends on the type of α subunit, the type of ß subunit and the presence of the γ subunit.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments.

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels. The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16. The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Modulatory Effects of Eschscholzia californica Alkaloids on Recombinant GABAA Receptors.

The California poppy (Eschscholzia californica Cham.) contains a variety of natural compounds including several alkaloids found exclusively in this plant. Because of the sedative, anxiolytic, and analgesic effects, this herb is currently sold in pharmacies in many countries. However, our understanding of these biological effects at the molecular level is still lacking. Alkaloids detected in E. californica could be hypothesized to act at GABAA receptors, which are widely expressed in the brain mainly at the inhibitory interneurons. Electrophysiological studies on a recombinant α 1 ß 2 γ 2 GABAA receptor showed no effect of N-methyllaurotetanine at concentrations lower than 30 µM. However, (S)-reticuline behaved as positive allosteric modulator at the α 3, α 5, and α 6 isoforms of GABAA receptors. The depressant properties of aerial parts of E. californica are assigned to chloride-current modulation by (S)-reticuline at the α 3 ß 2 γ 2 and α 5 ß 2 γ 2 GABAA receptors. Interestingly, α 1, α 3, and α 5 were not significantly affected by (R)-reticuline, 1,2-tetrahydroreticuline, codeine, and morphine-suspected (S)-reticuline metabolites in the rodent brain.

Flagellar membranes are rich in raft-forming phospholipids.

The observation that the membranes of flagella are enriched in sterols and sphingolipids has led to the hypothesis that flagella might be enriched in raft-forming lipids. However, a detailed lipidomic analysis of flagellar membranes is not available. Novel protocols to detach and isolate intact flagella from Trypanosoma brucei procyclic forms in combination with reverse-phase liquid chromatography high-resolution tandem mass spectrometry allowed us to determine the phospholipid composition of flagellar membranes relative to whole cells. Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells. In contrast, phosphatidylcholine and phosphatidylinositol are strongly depleted in flagella. Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella. Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the ß subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes.

Discovery of novel drug targets may lead to improved treatment of trypanosomiasis. We characterize here 2 gene products of Trypanosoma brucei that are essential for the growth of bloodstream form (BSF) parasites, as shown by RNA interference (RNAi)-mediated down-regulation of the individual mRNAs. The primary sequences of the 2 proteins--protein encoded by gene Tb927.1.4450 (TbK1) and protein encoded by gene Tb927.9.4820 (TbK2)--indicate that both belong to the family of putative, Ca(2+)-activated potassium channels. The proteins were expressed in Xenopus laevis oocytes and their functions investigated by use of electrophysiological techniques. Only combined expression of TbK1 and TbK2 results in the formation of sizeable currents, indicating that these proteins probably assemble into a heteromeric ion channel. The current mediated by this channel shows little time and voltage dependence and displays a permeability ratio of K(+)/Na(+) of >20. The known potassium channel blocker barium inhibits this channel with a half-maximal inhibitory concentration (IC50) of 98 ± 15 µM. The membrane potential of trypanosomes was measured with a fluorescent dye. Individual RNAi-mediated down-regulation of TbK1 or TbK2 eliminates a potassium conductance in the plasma membrane of BSF. Thus, this heteromeric potassium channel is involved in the modulation of the plasma membrane potential and represents a novel drug target in T. brucei.

Trypanosoma brucei Bloodstream Forms Depend upon Uptake of myo-Inositol for Golgi Complex Phosphatidylinositol Synthesis and Normal Cell Growth.

myo-Inositol is a building block for all inositol-containing phospholipids in eukaryotes. It can be synthesized de novo from glucose-6-phosphate in the cytosol and endoplasmic reticulum. Alternatively, it can be taken up from the environment via Na(+)- or H(+)-linked myo-inositol transporters. While Na(+)-coupled myo-inositol transporters are found exclusively in the plasma membrane, H(+)-linked myo-inositol transporters are detected in intracellular organelles. In Trypanosoma brucei, the causative agent of human African sleeping sickness, myo-inositol metabolism is compartmentalized. De novo-synthesized myo-inositol is used for glycosylphosphatidylinositol production in the endoplasmic reticulum, whereas the myo-inositol taken up from the environment is used for bulk phosphatidylinositol synthesis in the Golgi complex. We now provide evidence that the Golgi complex-localized T. brucei H(+)-linked myo-inositol transporter (TbHMIT) is essential in bloodstream-form T. brucei. Downregulation of TbHMIT expression by RNA interference blocked phosphatidylinositol production and inhibited growth of parasites in culture. Characterization of the transporter in a heterologous expression system demonstrated a remarkable selectivity of TbHMIT for myo-inositol. It tolerates only a single modification on the inositol ring, such as the removal of a hydroxyl group or the inversion of stereochemistry at a single hydroxyl group relative to myo-inositol.