Functional Expression In Xenopus Oocytes Research Articles

Activity, selection response and molecular mode of action of the isoxazoline afoxolaner in Tetranychus urticae.

Afoxolaner is a novel representative of the isoxazolines, a class of ectoparasiticides which has been commercialized for the control of tick and flea infestations in dogs. In this study, the biological efficacy of afoxolaner against the two-spotted spider mite Tetranychus urticae was evaluated. Furthermore, as isoxazolines are known inhibitors of γ-aminobutyric acid-gated chloride channels (GABACls), the molecular mode of action of afoxolaner on T.urticae GABACls (TuRdls) was studied using functional expression in Xenopus oocytes followed by two-electrode voltage-clamp (TEVC) electrophysiology, and results were compared with inhibition by fluralaner, fipronil and endosulfan. To examine the influence of known GABACl resistance mutations, H301A, I305T and A350T substitutions in TuRdl1 and a S301A substitution in TuRdl2 were introduced. Bioasassays revealed excellent efficacy of afoxolaner against all developmental stages and no cross-resistance was found in a panel of strains resistant to most currently used acaricides. Laboratory selection over a period of 3 years did not result in resistance. TEVC revealed clear antagonistic activity of afoxolaner and fluralaner for all homomeric TuRdl1/2/3 channels. The introduction of single, double or triple mutations to TuRdl1 and TuRdl2 did not lower channel sensitivity. By contrast, both endosulfan and fipronil had minimal antagonistic activities against TuRdl1/2/3, and channels carrying single mutations, whereas the sensitivity of double and triple TuRdl1 mutants was significantly increased. Our results demonstrate that afoxolaner is a potent antagonist of GABACls of T.urticae and has a powerful mode of action to control spider mites. © 2022 Society of Chemical Industry.

Agonist and antagonist properties of an insect GABA-gated chloride channel (RDL) are influenced by heterologous expression conditions.

Pentameric ligand-gated ion channels (pLGICs) activated by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) are expressed widely in both vertebrate and invertebrate species. One of the best characterised insect GABA-gated chloride channels is RDL, an abbreviation of ‘resistance to dieldrin’, that was originally identified by genetic screening in Drosophila melanogaster. Here we have cloned the analogous gene from the bumblebee Bombus terrestris audax (BtRDL) and examined its pharmacological properties by functional expression in Xenopus oocytes. Somewhat unexpectedly, the sensitivity of BtRDL to GABA, as measured by its apparent affinity (EC50), was influenced by heterologous expression conditions. This phenomenon was observed in response to alterations in the amount of cRNA injected; the length of time that oocytes were incubated before functional analysis; and by the presence or absence of a 3’ untranslated region. In contrast, similar changes in expression conditions were not associated with changes in apparent affinity with RDL cloned from D. melanogaster (DmRDL). Changes in apparent affinity with BtRDL were also observed following co-expression of a chaperone protein (NACHO). Similar changes in apparent affinity were observed with an allosteric agonist (propofol) and a non-competitive antagonist (picrotoxinin), indicating that expression-depended changes are not restricted to the orthosteric agonist binding site. Interestingly, instances of expression-dependent changes in apparent affinity have been reported previously for vertebrate glycine receptors, which are also members of the pLGIC super-family. Our observations with BtRDL are consistent with previous data obtained with vertebrate glycine receptors and indicates that agonist and antagonist apparent affinity can be influenced by the level of functional expression in a variety of pLGICs.

The novel isoxazoline ectoparasiticide lotilaner (Credelio\u2122): a non-competitive antagonist specific to invertebrates \u03b3-aminobutyric acid-gated chloride channels (GABACls)

BackgroundThe isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls). Lotilaner (Credelio™), a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties.MethodsIn this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the successful gene identification, cDNA cloning and functional expression in Xenopus oocytes of Drosohpila melanogaster (wild type and dieldrin/fipronil-resistant forms), Lepeophtheirus salmonis (an ectoparasite copepod crustacean of salmon), Rhipicephalus microplus and Canis lupus familiaris GABACls. Automated Xenopus oocyte two-electrode voltage clamp electrophysiology was used to assess GABACls functionality and to compare ion channel inhibition by lotilaner with that of established insecticides addressing GABACls as targets.ResultsIn these assays, we demonstrated that lotilaner is a potent non-competitive antagonist of insects (fly) GABACls. No cross-resistance with dieldrin or fipronil resistance mutations was detected, suggesting that lotilaner might bind to a site at least partly different from the one bound by known GABACl blockers. Using co-application experiments, we observed that lotilaner antagonism differs significantly from the classical open channel blocker fipronil. We finally confirmed for the first time that isoxazoline compounds are not only powerful antagonists of GABACls of acari (ticks) but also of crustaceans (sea lice), while no activity on a dog GABAA receptor was observed up to a concentration of 10 μM.ConclusionsTogether, these results demonstrate that lotilaner is a non-competitive antagonist specific to invertebrate’s γ-aminobutyric acid-gated chloride channels (GABACls). They contribute to our understanding of the mode of action of this new ectoparasiticide compound.

Five nicotinic acetylcholine receptor subunits from the Morotoge shrimp, Pandalopsis japonica: cloning, tissue distribution, and functional expression in Xenopus oocytes

The nicotinic acetylcholine receptor (nAChR) is a member of the ligand-gated ion channel (LGIC) family and is composed of five subunits arranged around a central pore. Expressed sequence tag screening and traditional cloning strategies revealed five full-length cDNAs encoding nAChR subunit homologs (Pajα3, Pajα10, Pajα11, Pajα12, and Pajβ1) in the Morotoge shrimp, Pandalopsis japonica. The nAChR subunits exhibited common structural characteristics, including a signal peptide sequence, a large N-terminal extracellular domain with conserved motifs for ligand binding (loops A–F), and a transmembrane (TM) domain with four hydrophobic TM motifs (TM1–TM4). Based on the conserved GEK motifs located just before TM2, all five nAChR subunits from P. japonica appear to be cation-selective ion channels. Among the five subunits, Pajα3 and Pajβ1 clustered together with insect core groups, whereas Pajα10, Pajα11, and Pajα12 were classified as a divergent group. Three distinct transcripts were identified in Pajα3, presumably due to alternative splicing between TM3 and TM4, which may be involved in channel formation with other subunits. All five nAChR subunits were expressed predominantly in neuronal tissues, including the brain, sinus gland/X-organ complex, thoracic ganglia, and abdominal ganglia, with no significant differences in subunit expression levels among the neuronal tissues. The five shrimp nAChR subunits could not be functionally expressed in Xenopus oocytes, but coexpression of Pajβ1 and rat α4 subunit (Rα4) formed functional channels responding to acetylcholine. Functional expression of vertebrate α subunit (Rα4) with invertebrate β1 subunit (Pajβ1) will expand our knowledge regarding the structural characteristics and molecular gating mechanism of invertebrate nAChRs.

The novel α7β2-nicotinic acetylcholine receptor subtype is expressed in mouse and human basal forebrain: biochemical and pharmacological characterization.

We examined α7β2-nicotinic acetylcholine receptor (α7β2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7β2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7β2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using β2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7β2- versus α7-nAChRs. This study represents the first direct confirmation of α7β2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7β2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7β2-nAChR subunit isoforms with different α7/β2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7β2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites.

Leaf Fructose Content Is Controlled by the Vacuolar Transporter SWEET17 in Arabidopsis

In higher plants, soluble sugars are mainly present as sucrose, glucose, and fructose. Sugar allocation is based on both source-to-sink transport and intracellular transport between the different organelles and depends on actual plant requirements. Under abiotic stress conditions, such as nitrogen limitation, carbohydrates accumulate in plant cells. Despite an increasing number of genetic studies, the genetic architecture determining carbohydrate composition is poorly known. Using a quantitative genetics approach, we determined that the carrier protein SWEET17 is a major factor controlling fructose content in Arabidopsis leaves. We observed that when SWEET17 expression is reduced, either by induced or natural variation, fructose accumulates in leaves, suggesting an enhanced storage capacity. Subcellular localization of SWEET17-GFP to the tonoplast and functional expression in Xenopus oocytes showed that SWEET17 is the first vacuolar fructose transporter to be characterized in plants. Physiological studies in planta provide evidence that SWEET17 acts to export fructose out of the vacuole. Overall, our results suggest that natural variation in leaf fructose levels is controlled by the vacuolar fructose transporter SWEET17. SWEET17 is highly conserved across the plant kingdom; thus, these findings offer future possibilities to modify carbohydrate partitioning in crops.

Functional expression in Xenopus oocytes reveals that human ferroportin is an iron exporter shared with zinc

Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Export of iron from enterocytes or macrophages to the blood is thought to be mediated by ferroportin (Fpn) under the control of hepcidin. Some mutations in Fpn result in macrophage iron loading whereas others, as a result of insensitivity to hepcidin, cause systemic iron overload. Whereas Fpn was identified a decade ago, there remains a paucity of information about how it works. We expressed GFP‐tagged human Fpn in RNA‐injected Xenopus oocytes and observed using confocal microscopy exclusive plasma‐membrane localization. As a first step in its characterization, we established an assay to detect functional expression of Fpn by microinjecting oocytes with 55Fe and measuring the 55Fe efflux over 30 min. Expression of Fpn increased the first‐order rate constants describing 55Fe efflux between 4‐fold and 100‐fold over control. Fpn‐mediated 55Fe efflux was maximal when the injectate contained nitrilotriacetic acid (NTA), and when we provided apotransferrin, bathophenanthroline disulfonate (BPS) and NTA in the incubation medium, whereas inclusion of ceruloplasmin had only a modest effect. Fpn‐mediated 55Fe was accelerated in pH 8.2 medium compared with pH 7.2 or pH 6.2 (P < 0.001). Fpn expression also stimulated the efflux of 65Zn (P < 0.001) but not of 54Mn, 64Cu or 109Cd (P > 0.11). We conclude that Fpn mediates cellular iron efflux and that Zn2+ counts among its narrow substrate profile.

Pentameric concatenated (α4)2(β2)3 and (α4)3(β2)2 nicotinic acetylcholine receptors: subunit arrangement determines functional expression

alpha4 and beta2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors. alpha4 and beta2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (alpha4)(2)(beta2)(3) or (alpha4)(3)(beta2)(2) receptors. Using two-electrode voltage-clamp techniques, we examined the ability of the concatenated constructs to produce functional expression in Xenopus oocytes. Functional constructs were further characterized in respect to agonists, competitive antagonists, Ca2+ permeability, sensitivity to modulation by Zn2+ and sensitivity to up-regulation by chaperone protein 14-3-3. We found that pentameric concatamers with a subunit arrangement of beta2_alpha4_beta2_alpha4_beta2 or beta2_alpha4_beta2_alpha4_alpha4 were stable and functional in Xenopus oocytes. By comparison, when alpha4 and beta2 were concatenated with a subunit order of beta2_beta2_alpha4_beta2_alpha4 or beta2_alpha4_alpha4_beta2_alpha4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both beta2_alpha4_beta2_alpha4_beta2 and beta2_alpha4_beta2_alpha4_alpha4 concatamers recapitulated the ACh concentration response curve, the sensitivity to Zn2+ modulation, Ca2+ permeability and the sensitivity to up-regulation by chaperone protein 14-3-3 of the corresponding non-linked (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors respectively. Using these concatamers, we found that most alpha4beta2-preferring compounds studied, including A85380, 5I-A85380, cytisine, epibatidine, TC2559 and dihydro-beta-erythroidine, demonstrate stoichiometry-specific potencies and efficacies. We concluded that the alpha4beta2 nicotinic ACh receptors produced with beta2_alpha4_beta2_alpha4_beta2 or beta2_alpha4_beta2_alpha4_alpha4 pentameric constructs are valid models of non-linked (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2) receptors respectively.

The Residues Determining Differences in Ion Affinities among the Alternative Splice Variants F, A, and B of the Mammalian Renal Na-K-Cl Cotransporter (NKCC2)

Three alternatively spliced variants of the renal Na-K-Cl cotransporter (NKCC2) are found in distinct regions of the thick ascending limb of the mammalian kidney; these variants mediate Na(+)K(+)2Cl(-) transport with different ion affinities. Here, we examine the specific residues involved in the variant-specific affinity differences, utilizing a mutagenic approach to change the NKCC2B variant into the A or F variant, with functional expression in Xenopus oocytes. The splice region contains the second transmembrane domain (TM2) and the putative intracellular loop (ICL1) connecting TM2 and TM3. It is found that the B variant is functionally changed to the F variant by replacement of six residues, half of the effect brought about by three TM2 residues and half by three ICL1 residues. The involvement of the ICL1 residues strongly suggests that this region of ICL1 may actually be part of a membrane-embedded domain. Changing six residues is also sufficient to bring about the smaller functional change from the B to the A variant; three residues in TM2 appear to be primarily responsible, two of which correspond to residues involved in the B-to-F changes. A B-variant mutation reported in a mild case of Bartter disease was found to render the cotransporter inactive. These results identify the combination of amino acid variations responsible for the differences among the three splice variants of NKCC2, and they support a model in which a reentrant loop following TM2 contributes to the chloride binding and translocation domains.

Specific Mutations in Transmembrane Helix 8 of Human Concentrative Na+/Nucleoside Cotransporter hCNT1 Affect Permeant Selectivity and Cation Coupling

The Na+/nucleoside cotransporters hCNT1 (650 residues) and hCNT2 (658 residues) are 72% identical in amino acid sequence and contain 13 putative transmembrane helices (TMs). Both transport uridine and adenosine but are otherwise selective for pyrimidine (system cit) and purine (system cif) nucleosides, respectively. Previously, we used site-directed mutagenesis and functional expression in Xenopus oocytes to identify two pairs of adjacent residues in TMs 7 and 8 of hCNT1 (Ser319-Gln320 and Ser353-Leu354) that, when converted to the corresponding residues in hCNT2 (Gly-Met and Thr-Val, respectively), changed the permeant selectivity of the transporter from cit to cif. We now report an investigation of the effects of corresponding mutations in TM 8 alone and demonstrate unique S353T- and L354V-induced changes in nucleoside specificity and cation coupling, respectively. hCNT1 mutation S353T produced a profound decrease in cytidine transport efficiency (Vmax/Km ratio) and, in combination with L354V (S353T/L354V), resulted in a novel uridine-preferring transport phenotype. In addition, the L354V mutation markedly increased the apparent affinity of hCNT1 for Na+ and Li+. Both hCNT1 TM 8 residues exhibited uridine-protectable inhibition by p-chloromercuribenzene sulfonate when converted to Cys, suggesting that they occupy positions within or closely adjacent to a common cation/nucleoside translocation pore.

Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater

Sulfate is required for proper cell growth and development of all organisms. We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration.

Functional Expression in Xenopus Oocytes of Gap-junctional Hemichannels Formed by a Cysteine-less Connexin 43

Gap-junctional channels are formed by two connexons or gap-junctional hemichannels in series, with each connexon conformed by six connexin molecules. As with other membrane proteins, structural information on connexons can potentially be obtained with techniques that take advantage of the highly specific thiol chemistry by positioning Cys residues at locations of interest, ideally in an otherwise Cys-less protein. It has been shown that conserved Cys residues located in the extracellular loops of connexins are essential for the docking of connexons from adjacent cells, preventing the formation of functional gap-junctional channels. Here we engineered a Cys-less version of connexin 43 (Cx43) and assessed its function using a Xenopus oocyte expression system. The Cys-less protein was expressed at the plasma membrane and did not form gap-junctional channels but formed hemichannels that behave similarly to those formed by Cx43 in terms of permeation to carboxyfluorescein. The carboxyfluorescein permeability of Cys-less hemichannels was increased by protein kinase C inhibition, like the wild-type Cx43 hemichannels. We generated a protein with a single Cys in a position (residue 34) thought to face the channel pore and show that thiol modification of the Cys abolishes the carboxyfluorescein permeability. We conclude that Cysless Cx43 forms regulated functional hemichannels, and therefore Cys-less Cx43 is a useful tool for future structural studies.

Rapid Report

Coexpression of KCNQ2 and KCNQ3 channels results in a 10‐fold increased current amplitude compared to that of KCNQ2 alone, suggesting the formation of heteromultimeric channels. There is no interaction of either channel with KCNQ1. We evaluated the C‐terminus as a potential interaction domain by construction of chimeras with interchanged C‐termini of KCNQ1, KCNQ2 and KCNQ3 and functional expression in Xenopus oocytes. The chimera of KCNQ1 with a KCNQ2 C‐terminus (Q1ctQ2) showed an 8‐fold increase in current amplitude, and Q1ctQ3 a 3‐fold increase when coexpressed with KCNQ3 and KCNQ2, respectively, indicating that the C‐terminus contains an interaction domain. To characterize this interacting region, we studied further chimeras of KCNQ1 containing different parts of the KCNQ3 C‐terminus for interaction with KCNQ2. We also evaluated short sequences of the KCNQ2 C‐terminus for a dominant‐negative effect on Q1ctQ3. According to the results of these experiments, functional interaction of KCNQ2 and KCNQ3 requires a highly conserved region of about 80 amino acids, previously called the A‐domain, plus either 40 residues downstream of the A‐domain (B‐domain) or the proximal C‐terminus between S6 and the A‐domain. Furthermore, the chimeras Q1ctQ3 and Q2ctQ3 showed > 10‐fold increased current amplitudes compared to KCNQ1 or KCNQ2 alone and a strong depolarizing shift of voltage‐dependent activation. The proximal part of the KCNQ3 C‐terminus was necessary to produce these effects. Our results indicate that specific parts of the C‐terminus enable the interaction between KCNQ2 and KCNQ3 channels and that different parts of the KCNQ3 C‐terminus are important for regulating current amplitude.

C-terminal interaction of KCNQ2 and KCNQ3 K+ channels.

Coexpression of KCNQ2 and KCNQ3 channels results in a 10-fold increased current amplitude compared to that of KCNQ2 alone, suggesting the formation of heteromultimeric channels. There is no interaction of either channel with KCNQ1. We evaluated the C-terminus as a potential interaction domain by construction of chimeras with interchanged C-termini of KCNQ1, KCNQ2 and KCNQ3 and functional expression in Xenopus oocytes. The chimera of KCNQ1 with a KCNQ2 C-terminus (Q1ctQ2) showed an 8-fold increase in current amplitude, and Q1ctQ3 a 3-fold increase when coexpressed with KCNQ3 and KCNQ2, respectively, indicating that the C-terminus contains an interaction domain. To characterize this interacting region, we studied further chimeras of KCNQ1 containing different parts of the KCNQ3 C-terminus for interaction with KCNQ2. We also evaluated short sequences of the KCNQ2 C-terminus for a dominant-negative effect on Q1ctQ3. According to the results of these experiments, functional interaction of KCNQ2 and KCNQ3 requires a highly conserved region of about 80 amino acids, previously called the A-domain, plus either 40 residues downstream of the A-domain (B-domain) or the proximal C-terminus between S6 and the A-domain. Furthermore, the chimeras Q1ctQ3 and Q2ctQ3 showed > 10-fold increased current amplitudes compared to KCNQ1 or KCNQ2 alone and a strong depolarizing shift of voltage-dependent activation. The proximal part of the KCNQ3 C-terminus was necessary to produce these effects. Our results indicate that specific parts of the C-terminus enable the interaction between KCNQ2 and KCNQ3 channels and that different parts of the KCNQ3 C-terminus are important for regulating current amplitude.

Cloning, functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k).

The nitrate transporter from Chlorella sorokiniana (accession number AY026523) has been cloned by screening a cDNA library based on mRNA isolated after 30 min treatment of Chlorella with 5 mM nitrate and with a RT-PCR product (730 bp) as a probe. The Chlorella sequence has similarity to known nitrate transporters of the NRT2 family (high-affinity nitrate transporters). The cDNA clone was used for functional expression in Xenopus oocytes and a nitrate-dependent current was measured at pH 5.5 but not at pH 7.4. A second algal gene or a second gene product was not needed for functional expression in Xenopus. Inhibitor studies in Chlorella indicated that protein phosphorylation/dephosphorylation is involved in nitrate induction of ChNRT2.1. In addition to nitrate, ChNRT2.1 expression is induced by nitroprusside, a NO donor, and is affected by glucose.

Outer pore residues control the H(+) and K(+) sensitivity of the Arabidopsis potassium channel AKT3.

The Arabidopsis phloem channel AKT3 is the founder of a subfamily of shaker-like plant potassium channels characterized by weak rectification, Ca(2+) block, proton inhibition, and, as shown in this study, K(+) sensitivity. In contrast to inward-rectifying, acid-activated K(+) channels of the KAT1 family, extracellular acidification decreases AKT3 currents at the macroscopic and single-channel levels. Here, we show that two distinct sites within the outer mouth of the K(+)-conducting pore provide the molecular basis for the pH sensitivity of this phloem channel. After generation of mutant channels and functional expression in Xenopus oocytes, we identified the His residue His-228, which is proximal to the K(+) selectivity filter (GYGD) and the distal Ser residue Ser-271, to be involved in proton susceptibility. Mutations of these sites, H228D and S271E, drastically reduced the H(+) and K(+) sensitivity of AKT3. Although in K(+)-free bath solutions outward K(+) currents were abolished completely in wild-type AKT3, S271E as well as the AKT3-HDSE double mutant still mediated K(+) efflux. We conclude that the pH- and K(+)-dependent properties of the AKT3 channel involve residues in the outer mouth of the pore. Both properties, H(+) and K(+) sensitivity, allow the fine-tuning of the phloem channel and thus seem to represent important elements in the control of membrane potential and sugar loading.

Characterization of the human fMLP receptor in neutrophils and in Xenopus oocytes

1. N-formyl peptides (e.g. fMLP; N-formyl-L-methionyl-L-leucyl-phenylalanine) are potent mediators for inflammatory reactions. We report functional expression in Xenopus oocytes of human fMLP-R98 cDNA, without co-expression of the promiscuous G-protein subunit, Galpha-16. 2. Stimulation of voltage-clamped oocytes (-70 mV) with fMLP produced a dose-dependent biphasic inward current with fast and slow components. Analysis using GTP-gamma-S and cholera and pertussis toxins suggested these currents are mediated by an endogenous G-protein of the Gq family. 3. The fast current reversed at -25 mV and was blocked by SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid), suggesting the current is carried by Cl(-). The slow current showed weak inward rectification, was Ca(2+)-dependent and blocked by Cd(2+), 4-AP (4-aminopyridine) and haloperidol, suggesting activation of a mixed population of cation channels. 4. Comparative experiments with human neutrophils using flow cytometric analysis showed that the proportion of neutrophils activated by fMLP was reduced in the presence of SITS, in the absence of external calcium and in the presence of Cd(2+), TEA (tetraethylammonium) and haloperidol but not 4-AP. In addition, the oxidative burst from activated neutrophils was reduced by SITS and by the absence of external calcium but not by Cd(2+), TEA, 4-AP or haloperidol. 5. We suggest that in human neutrophils activation by fMLP is dependent on store-operated calcium influx that appears to be regulated by Cl(-) channels and linked, in part, to non-selective cation channels.

Structural, Functional, and Evolutionary Characterization of Novel Members of the Allatostatin Receptor Family from Insects

By using degenerate primers based on known mammalian somatostatin receptors and the recently identified Drosophila allatostatin receptors (AlstR), we have cloned a novel receptor for the neuropeptide, allatostatin, from the cockroach Periplaneta americana. The receptor exhibits about 60% amino acid identity in the transmembrane regions when compared to the two known AlstRs from Drosophila melanogaster. In addition, two cDNA fragments were obtained from the stick insect Carausius morosus, one of which is similar to Drosophila AlstR, whereas the other is more similar to mammalian somatostatin receptors. Functional expression in Xenopus oocytes shows that the Periplaneta-AlstR exhibits high affinity to endogenous cockroach allatostatin peptides. Studies with synthetic peptides demonstrate that agonistic activity is mediated by the conserved C-terminal pentapeptide YXFGL-amide; in this sequence, amidation of the C-terminus is obligatory to maintain affinity. Thus, our studies provide a molecular basis for understanding the widespread biological activities of the allatostatin peptides.

Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum requires glucose as its energy source to multiply within erythrocytes but is separated from plasma by multiple membrane systems. The mechanism of delivery of substrates such as glucose to intraerythrocytic parasites is unclear. We have developed a system for robust functional expression in Xenopus oocytes of the P. falciparum asexual stage hexose permease, PfHT1, and have analyzed substrate specificities of PfHT1. We show that PfHT1 (a high-affinity glucose transporter, K(m) approximately 1.0 mM) also transports fructose (K(m) approximately 11.5 mM). Fructose can replace glucose as an energy source for intraerythrocytic parasites. PfHT1 binds fructose in a furanose conformation and glucose in a pyranose form. Fructose transport by PfHT1 is ablated by mutation of a single glutamine residue, Q169, which is predicted to lie within helix 5 of the hexose permeation pathway. Glucose transport in the Q169N mutant is preserved. Comparison in oocytes of transport properties of PfHT1 and human facilitative glucose transporter (GLUT)1, an archetypal mammalian hexose transporter, combined with studies on cultured P. falciparum, has clarified hexose permeation pathways in infected erythrocytes. Glucose and fructose enter erythrocytes through separate permeation pathways. Our studies suggest that both substrates enter parasites via PfHT1.

Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy.

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (K(m) 0.32+/-0.05 mM) than the pyrimidine nucleoside uridine (K(m) 3.5+/-1.1 mM). It also efficiently transports nucleobases such as adenine (K(m) 0.32+/-0.10 mM) and hypoxanthine (K(m) 0.41+/-0.1 mM), and anti-viral 3'-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR [6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, or nitrobenzylthioinosine] and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.