Functional Characterization Supports Multiple Evolutionary Origins of Pheromone Receptors in Bark Beetles.

Chemical communication using pheromones is thought to have contributed to the diversification and speciation of insects. The species-specific pheromones are detected by specialized pheromone receptors (PRs). Whereas the evolution and function of PRs have been extensively studied in Lepidoptera, only a few PRs have been identified in beetles, which limits our understanding of their evolutionary histories and physiological functions. To shed light on these questions, we aimed to functionally characterize potential PRs in the spruce bark beetle Ips typographus ("Ityp") and explore their evolutionary origins and molecular interactions with ligands. Males of this species release an aggregation pheromone comprising 2-methyl-3-buten-2-ol and (4S)-cis-verbenol, which attracts both sexes to attacked trees. Using two systems for functional characterization, we show that the highly expressed odorant receptor (OR) ItypOR41 responds specifically to (4S)-cis-verbenol, with structurally similar compounds eliciting minor responses. We next targeted the closely related ItypOR40 and ItypOR45. Whereas ItypOR40 was unresponsive, ItypOR45 showed an overlapping response profile with ItypOR41, but a broader tuning. Our phylogenetic analysis shows that these ORs are present in a different OR clade as compared to all other known beetle PRs, suggesting multiple evolutionary origins of PRs in bark beetles. Next, using computational analyses and experimental validation, we reveal two amino acid residues (Gln179 and Trp310) that are important for ligand binding and pheromone specificity of ItypOR41 for (4S)-cis-verbenol, possibly via hydrogen bonding to Gln179. Collectively, our results shed new light on the origins, specificity, and ligand binding mechanisms of PRs in beetles.

Effect of Synthetic Vitreous Fiber Exposure on TMEM16A Channels in a Xenopus laevis Oocyte Model.

Many years ago, asbestos fibers were banned and replaced by synthetic vitreous fibers because of their carcinogenicity. However, the toxicity of the latter fibers is still under debate, especially when it concerns the early fiber interactions with biological cell membranes. Here, we aimed to investigate the effects of a synthetic vitreous fiber named FAV173 on the Xenopus laevis oocyte membrane, the cell model we have already used to characterize the effect of crocidolite asbestos fiber exposure. Using an electrophysiological approach, we found that, similarly to crocidolite asbestos, FAV173 was able to stimulate a chloride outward current evoked by step membrane depolarizations, that was blocked by the potent and specific TMEM16A channel antagonist Ani9. Exposure to FAV173 fibers also altered the oocyte cell membrane microvilli morphology similarly to crocidolite fibers, most likely as a consequence of the TMEM16A protein interaction with actin. However, FAV173 only partially mimicked the crocidolite fibers effects, even at higher fiber suspension concentrations. As expected, the crocidolite fibers' effect was more similar to that induced by the co-treatment with (Fe3+ + H2O2), since the iron content of asbestos fibers is known to trigger reactive oxygen species (ROS) production. Taken together, our findings suggest that FAV173 may be less harmful that crocidolite but not ineffective in altering cell membrane properties.

A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts.

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.

Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis.

BACKGROUND: Variants in SLC34A2 encoding the sodium-dependent phosphate transport protein 2b (NaPi-IIb) cause the rare lung disease pulmonary alveolar microlithiasis (PAM). PAM is characterised by the deposition of calcium-phosphate concretions in the alveoli usually progressing over time. No effective treatment is available. So far, 30 allelic variants in patients have been reported but only a few have been functionally characterised. This study aimed to determine the impact of selected SLC34A2 variants on transporter expression and phosphate uptake in cellular studies. METHODS: Two nonsense variants (c.910A > T and c.1456C > T), one frameshift (c.1328delT), and one in-frame deletion (c.1402_1404delACC) previously reported in patients with PAM were selected for investigation. Wild-type and mutant c-Myc-tagged human NaPi-IIb constructs were expressed in Xenopus laevis oocytes. The transport function was investigated with a 32Pi uptake assay. NaPi-IIb protein expression and localisation were determined with immunoblotting and immunohistochemistry, respectively. RESULTS: Oocytes injected with the wild-type human NaPi-IIb construct had significant 32Pi transport compared to water-injected oocytes. In addition, the protein had a molecular weight as expected for the glycosylated form, and it was readily detectable in the oocyte membrane. Although the protein from the Thr468del construct was synthesised and expressed in the oocyte membrane, phosphate transport was similar to non-injected control oocytes. All other mutants were non-functional and not expressed in the membrane, consistent with the expected impact of the truncations caused by premature stop codons. CONCLUSIONS: Of four analysed SLC34A2 variants, only the Thr468del showed similar protein expression as the wild-type cotransporter in the oocyte membrane. All mutant transporters were non-functional, supporting that dysfunction of NaPi-IIb underlies the pathology of PAM.

Xenopus laevis Oocyte Array Fluidic Device Integrated with Microelectrodes for A Compact Two-Electrode Voltage Clamping System.

We report on a compact two-electrode voltage clamping system composed of microfabricated electrodes and a fluidic device for Xenopus laevis oocytes. The device was fabricated by assembling Si-based electrode chips and acrylic frames to form fluidic channels. After the installation of Xenopus oocytes into the fluidic channels, the device can be separated in order to measure changes in oocyte plasma membrane potential in each channel using an external amplifier. Using fluid simulations and experiments, we investigated the success rates of Xenopus oocyte arrays and electrode insertion with respect to the flow rate. We successfully located each oocyte in the array and detected oocyte responses to chemical stimuli using our device.

Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters.

The high-affinity/low-capacity system Slc15a2 (PepT2) is responsible for the reuptake of di/tripeptides from the renal proximal tubule, but it also operates in many other tissues and organs. Information regarding PepT2 in teleost fish is limited and, to date, functional data are available from the zebrafish (Danio rerio) only. Here, we report the identification of two slc15a2 genes in the Atlantic salmon (Salmo salar) genome, namely slc15a2a and slc15a2b. The two encoded PepT2 proteins share 87% identity and resemble both structurally and functionally the canonical vertebrate PepT2 system. The mRNA tissue distribution analyses reveal a widespread distribution of slc15a2a transcripts, being more abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and the distal part of the gastrointestinal tract. The function of the two transporters was investigated by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp recordings of transport and presteady-state currents. Both PepT2a and PepT2b in the presence of Gly-Gln elicit pH-dependent and Na+ independent inward currents. The biophysical and kinetic analysis of the recorded currents defined the transport properties, confirming that the two Atlantic salmon PepT2 proteins behave as high-affinity/low-capacity transporters. The recent structures and the previous kinetic schemes of rat and human PepT2 qualitatively account for the characteristics of the two Atlantic salmon proteins. This study is the first to report on the functional expression of two PepT2-type transporters that operate in the same vertebrate organism as a result of (a) gene duplication process(es). KEY POINTS: Two slc15a2-type genes, slc15a2a and slc15a2b coding for PepT2-type peptide transporters were found in the Atlantic salmon. slc15a2a transcripts, widely distributed in the fish tissues, are abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and distal gastrointestinal tract. Amino acids involved in vertebrate Slc15 transport function are conserved in PepT2a and PepT2b proteins. Detailed kinetic analysis indicates that both PepT2a and PepT2b operate as high-affinity transporters. The kinetic schemes and structures proposed for the mammalian models of PepT2 are suitable to explain the function of the two Atlantic salmon transporters.

Two adjacent phosphorylation sites in the C-terminus of the channel's α-subunit have opposing effects on epithelial sodium channel (ENaC) activity.

How phosphorylation of the epithelial sodium channel (ENaC) contributes to its regulation is incompletely understood. Previously, we demonstrated that in outside-out patches ENaC activation by serum- and glucocorticoid-inducible kinase isoform 1 (SGK1) was abolished by mutating a serine residue in a putative SGK1 consensus motif RXRXX(S/T) in the channel's α-subunit (S621 in rat). Interestingly, this serine residue is followed by a highly conserved proline residue rather than by a hydrophobic amino acid thought to be required for a functional SGK1 consensus motif according to in vitro data. This suggests that this serine residue is a potential phosphorylation site for the dual-specificity tyrosine phosphorylated and regulated kinase 2 (DYRK2), a prototypical proline-directed kinase. Its phosphorylation may prime a highly conserved preceding serine residue (S617 in rat) to be phosphorylated by glycogen synthase kinase 3 ß (GSK3ß). Therefore, we investigated the effect of DYRK2 on ENaC activity in outside-out patches of Xenopus laevis oocytes heterologously expressing rat ENaC. DYRK2 included in the pipette solution significantly increased ENaC activity. In contrast, GSK3ß had an inhibitory effect. Replacing S621 in αENaC with alanine (S621A) abolished the effects of both kinases. A S617A mutation reduced the inhibitory effect of GKS3ß but did not prevent ENaC activation by DYRK2. Our findings suggest that phosphorylation of S621 activates ENaC and primes S617 for subsequent phosphorylation by GSK3ß resulting in channel inhibition. In proof-of-concept experiments, we demonstrated that DYRK2 can also stimulate ENaC currents in microdissected mouse distal nephron, whereas GSK3ß inhibits the currents.

Different efficiency of auxiliary/chaperone proteins to promote the functional reconstitution of honeybee glutamate and acetylcholine receptors in Xenopus laevis oocytes.

Heterologous expression systems (e.g., Xenopus laevis oocytes) are useful to study the biophysical properties and pharmacology of ionotropic receptors such as ionotropic glutamate (iGLuRs) and nicotinic acetylcholine (nAChRs) receptors. However, insect receptors often require the co-expression of chaperone proteins to be functional. Only few iGluRs and nAChRs have been successfully expressed in such systems. Here, we compared the efficiency of chaperone proteins to promote the functional expression of one Apis mellifera iGluR and several nAChR subunit combinations (α1α8ß1, α7, α2α8ß1 and α2α7α8ß1) in Xenopus oocytes. To this end, we cloned a new iGluR (GluR-1) and potential chaperone proteins (e.g., SOL-1, Neto, NACHO) and tested more than 40 combinations of human, nematode and honeybee proteins. We obtained robust expression of GluR-1 and α1α8ß1 when co-expressed with honeybee chaperone proteins and found that nAChR expression critically depended on the α1 subunit N-terminal sequence. We recorded small ACh-gated currents in few oocytes when the α7 subunit was co-expressed with Caenorhabditis elegans RIC-3, but none of the chaperone proteins allowed efficient expression of α2α8ß1 or α2α7α8ß1. Our results show that only some protein combinations can reconstitute functional receptors in Xenopus oocytes and that protein combination efficient in one species is not always efficient in another species.

The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure-Function Relationships in Mammalian SLC6 Transporters.

To the SLC6 family belong 20 human transporters that utilize the sodium electrochemical gradient to move biogenic amines, osmolytes, amino acids and related compounds into cells. They are classified into two functional groups, the Neurotransmitter transporters (NTT) and Nutrient amino acid transporters (NAT). Here we summarize how since their first cloning in 1998, the insect (Lepidopteran) Orthologs of the SLC6 family transporters have represented very important tools for investigating functional-structural relationships, mechanism of transport, ion and pH dependence and substate interaction of the mammalian (and human) counterparts.

Characterization of erenumab and rimegepant on calcitonin gene-related peptide induced responses in Xenopus Laevis oocytes expressing the calcitonin gene-related peptide receptor and the amylin-1 receptor.

BACKGROUND: The clinical use of calcitonin gene-related peptide receptor (CGRP-R) antagonists and monoclonal antibodies against CGRP and CGRP-R has offered new treatment possibilities for migraine patients. CGRP activates both the CGRP-R and structurally related amylin 1 receptor (AMY1-R). The relative effect of erenumab and the small-molecule CGRP-R antagonist, rimegepant, towards the CGRP-R and AMY-R needs to be further characterized. METHODS: The effect of CGRP and two CGRP-R antagonists were examined in Xenopus laevis oocytes expressing human CGRP-R, human AMY1-R and their subunits. RESULTS: CGRP administered to receptor expressing oocytes induced a concentration-dependent increase in current with the order of potency CGRP-R> > AMY1-R > calcitonin receptor (CTR). There was no effect on single components of the CGRP-R; calcitonin receptor-like receptor and receptor activity-modifying protein 1. Amylin was only effective on AMY1-R and CTR. Inhibition potencies (pIC50 values) for erenumab on CGRP induced currents were 10.86 and 9.35 for CGRP-R and AMY1-R, respectively. Rimegepant inhibited CGRP induced currents with pIC50 values of 11.30 and 9.91 for CGRP-R and AMY1-R, respectively. CONCLUSION: Our results demonstrate that erenumab and rimegepant are potent antagonists of CGRP-R and AMY1-R with 32- and 25-times preference for the CGRP-R over the AMY1-R, respectively. It is discussed if this difference in affinity between the two receptors is the likely reason why constipation is a common and serious adverse effect during CGRP-R antagonism but less so with CGRP binding antibodies.

Identification and characterization of the Atlantic salmon peptide transporter 1a.

Peptide transporter 1 (PepT1) mediates the uptake of dietary di-/tripeptides in vertebrates. However, in teleost fish gut, more than one PepT1-type transporter might operate, because of teleost-specific whole gen(om)e duplication event(s) that occurred during evolution. Here, we describe a novel teleost di-/tripeptide transporter, i.e., the Atlantic salmon (Salmo salar) peptide transporter 1a [PepT1a; or solute carrier family 15 member 1a (Slc15a1a)], which is a paralog (77% similarity and 64% identity at the amino acid level) of the well-described Atlantic salmon peptide transporter 1b [PepT1b, alias PepT1; or solute carrier family 15 member 1b (Slc15a1b)]. Comparative analysis and evolutionary relationships of gene/protein sequences were conducted after ad hoc database mining. Tissue mRNA expression analysis was performed by quantitative real-time PCR, whereas transport function analysis was accomplished by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp measurements. Atlantic salmon pept1a is highly expressed in the proximal intestine (pyloric ceca ≈ anterior midgut > midgut >> posterior midgut), in the same gut regions as pept1b but notably ~5-fold less abundant. Like PepT1b, Atlantic salmon PepT1a is a low-affinity/high-capacity system. Functional analysis showed electrogenic, Na+-independent/pH-dependent transport and apparent substrate affinity (K0.5) values for Gly-Gln of 1.593 mmol/L at pH 7.6 and 0.076 mmol/L at pH 6.5. In summary, we show that a piscine PepT1a-type transporter is functional. Defining the role of Atlantic salmon PepT1a in the gut will help to understand the evolutionary and functional relationships among peptide transporters. Its functional characterization will contribute to elucidate the relevance of peptide transporters in Atlantic salmon nutritional physiology.

Cav2.1 C-terminal fragments produced in Xenopus laevis oocytes do not modify the channel expression and functional properties.

The sequence and genomic organization of the CACNA1A gene that encodes the Cav2.1 subunit of both P and Q-type Ca2+ channels are well conserved in mammals. In human, rat and mouse CACNA1A, the use of an alternative acceptor site at the exon 46-47 boundary results in the expression of a long Cav2.1 splice variant. In transfected cells, the long isoform of human Cav2.1 produces a C-terminal fragment, but it is not known whether this fragment affects Cav2.1 expression or functional properties. Here, we cloned the long isoform of rat Cav2.1 (Cav2.1(e47)) and identified a novel variant with a shorter C-terminus (Cav2.1(e47s)) that differs from those previously described in the rat and mouse. When expressed in Xenopus laevis oocytes, Cav2.1(e47) and Cav2.1(e47s) displayed similar functional properties as the short isoform (Cav2.1). We show that Cav2.1 isoforms produced short (CT1) and long (CT1(e47)) C-terminal fragments that interacted in vivo with the auxiliary Cavß4a subunit. Overexpression of the C-terminal fragments did not affect Cav2.1 expression and functional properties. Furthermore, the functional properties of a Cav2.1 mutant without the C-terminal Cavß4 binding domain (Cav2.1ΔCT2) were similar to those of Cav2.1 and were not influenced by the co-expression of the missing fragments (CT2 or CT2(e47)). Our results exclude a functional role of the C-terminal fragments in Cav2.1 biophysical properties in an expression system widely used to study this channel.

Human cationic amino acid transporters are not affected by direct nitros(yl)ation.

A direct inhibiting effect of NO on the function of CAT-1 and -2A has been postulated to occur via nitrosylation of cysteine residues in the transporters. Neither the NO donor SNAP nor a mixture of SIN-1 and Spermine NONOate, that generates the strong nitrosating agent N2O3, reduced CAT-mediated L-arginine transport. Direct nitros(yl)ation does either not occur in CATs or does not affect their transport function. A regulatory effect of NO or nitrosating agents on CAT-mediated transport under physiological conditions seems, therefore, unlikely.

Functional differences in transport properties of natural HKT1;1 variants influence shoot Na+ exclusion in grapevine rootstocks.

Under salinity, Vitis spp. rootstocks can mediate salt (NaCl) exclusion from grafted V. vinifera scions enabling higher grapevine yields and production of superior wines with lower salt content. Until now, the genetic and mechanistic elements controlling sodium (Na+ ) exclusion in grapevine were unknown. Using a cross between two Vitis interspecific hybrid rootstocks, we mapped a dominant quantitative trait locus (QTL) associated with leaf Na+ exclusion (NaE) under salinity stress. The NaE locus encodes six high-affinity potassium transporters (HKT). Transcript profiling and functional characterization in heterologous systems identified VisHKT1;1 as the best candidate gene for controlling leaf Na+ exclusion. We characterized four proteins encoded by unique VisHKT1;1 alleles from the parents, and revealed that the dominant HKT variants exhibit greater Na+ conductance with less rectification than the recessive variants. Mutagenesis of VisHKT1;1 and TaHKT1.5-D from bread wheat, demonstrated that charged amino acid residues in the eighth predicted transmembrane domain of HKT proteins reduces inward Na+ conductance, and causes inward rectification of Na+ transport. The origin of the recessive VisHKT1;1 alleles was traced to V. champinii and V. rupestris. We propose that the genetic and functional data presented here will assist with breeding Na+ -tolerant grapevine rootstocks.

Identification of a mammalian silicon transporter.

Silicon (Si) has long been known to play a major physiological and structural role in certain organisms, including diatoms, sponges, and many higher plants, leading to the recent identification of multiple proteins responsible for Si transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding about the biochemical pathways that enable Si homeostasis. Here we report the identification of a mammalian efflux Si transporter, namely Slc34a2 (also termed NaPiIIb), a known sodium-phosphate cotransporter, which was upregulated in rat kidney following chronic dietary Si deprivation. Normal rat renal epithelium demonstrated punctate expression of Slc34a2, and when the protein was heterologously expressed in Xenopus laevis oocytes, Si efflux activity (i.e., movement of Si out of cells) was induced and was quantitatively similar to that induced by the known plant Si transporter OsLsi2 in the same expression system. Interestingly, Si efflux appeared saturable over time, but it did not vary as a function of extracellular [Formula: see text] or Na+ concentration, suggesting that Slc34a2 harbors a functionally independent transport site for Si operating in the reverse direction to the site for phosphate. Indeed, in rats with dietary Si depletion-induced upregulation of transporter expression, there was increased urinary phosphate excretion. This is the first evidence of an active Si transport protein in mammals and points towards an important role for Si in vertebrates and explains interactions between dietary phosphate and silicon.

Maitotoxin Is a Potential Selective Activator of the Endogenous Transient Receptor Potential Canonical Type 1 Channel in Xenopus laevis Oocytes.

Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.

Amino acid transporter B(0)AT1 (slc6a19) and ancillary protein: impact on function.

Amino acids play an important role in the metabolism of all organisms. Their epithelial re-absorption is due to specific transport proteins, such as B(0)AT1, a Na(+)-coupled neutral amino acid symporter belonging to the solute carrier 6 family. Here, a recently cloned fish orthologue, from the intestine of Salmo salar, was electrophysiologically characterized with the two-electrode voltage clamp technique, in Xenopus laevis oocytes heterologously expressing the transporter. Substrate specificity, apparent affinities and the ionic dependence of the transport mechanism were determined in the presence of specific collectrin. Results demonstrated that like the human, but differently from sea bass (Dicentrarchus labrax) orthologue, salmon B(0)AT1 needs to be associated with partner proteins to be correctly expressed at the oocyte plasma membrane. Cloning of sea bass collectrin and comparison of membrane expression and functionality of the B(0)AT1 orthologue transporters allowed a deeper investigation on the role of their interactions. The parameters acquired by electrophysiological and immunolocalization experiments in the mammalian and fish transporters contributed to highlight the dynamic of relations and impacts on transport function of the ancillary proteins. The comparative characterization of the physiological parameters of amino acid transporters with auxiliary proteins can help the comprehension of the regulatory mechanism of essential nutrient absorption.

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.

Functional characterization of the human facilitative glucose transporter 12 (GLUT12) by electrophysiological methods.

GLUT12 is a member of the facilitative family of glucose transporters. The goal of this study was to characterize the functional properties of GLUT12, expressed in Xenopus laevis oocytes, using radiotracer and electrophysiological methods. Our results showed that GLUT12 is a facilitative sugar transporter with substrate selectivity: d-glucose ≥ α-methyl-d-glucopyranoside (α-MG) > 2-deoxy-d-glucose(2-DOG) > d-fructose = d-galactose. α-MG is a characteristic substrate of the Na(+)/glucose (SGLT) family and has not been shown to be a substrate of any of the GLUTs. In the absence of sugar, (22)Na(+) was transported through GLUT12 at a higher rate (40%) than noninjected oocytes, indicating that there is a Na(+) leak through GLUT12. Genistein, an inhibitor of GLUT1, also inhibited sugar uptake by GLUT12. Glucose uptake was increased by the PKA activator 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) but not by the PKC activator phorbol-12-myristate-13-acetate (PMA). In high K(+) concentrations, glucose uptake was blocked. Addition of glucose to the external solution induced an inward current with a reversal potential of approximately -15 mV and was blocked by Cl(-) channel blockers, indicating the current was carried by Cl(-) ions. The sugar-activated Cl(-) currents were unaffected by genistein. In high external K(+) concentrations, sugar-activated Cl(-) currents were also blocked, indicating that GLUT12 activity is voltage dependent. Furthermore, glucose-induced current was increased by the PKA activator 8-Br-cAMP but not by the PKC activator PMA. These new features of GLUT12 are very different from those described for other GLUTs, indicating that GLUT12 must have a specific physiological role within glucose homeostasis, still to be discovered.

ANP and CNP activate CFTR expressed in Xenopus laevis oocytes by direct activation of PKA.

CONTEXT: Acting through different receptors, natriuretic peptides (atrial natriuretic peptide [ANP], brain type natriuretic peptide [BNP] and C-type natriuretic peptide [CNP]) increase intracellular cGMP, which then stimulates different pathways that activate fluid secretion. OBJECTIVE: We used two-electrode voltage clamping to define the dominant pathway that is employed when natriuretic peptides activate cystic fibrosis transmembrane conductance regulator (CFTR) in the Xenopus oocyte expression system. Natriuretic peptides could activate CFTR by 1) cGMP cross-activation of protein kinase A (PKA), 2) cGMP activation of cGMP-dependent protein kinase II, 3) cGMP inhibition of phosphodiesterase type III (PDE3), or 4) direct activation of CFTR. MATERIALS AND METHODS: cRNA-microinjected Xenopus laevis oocytes were perfused with diverse compounds that examined these pathways of natriuretic peptide signaling. RESULTS AND DISCUSSION: ANP stimulated the shark CFTR (sCFTR)-mediated chloride conductance and this activation was inhibited by H-89, a specific inhibitor of PKA. After co-expression of the CNP receptor (NPR-B), sCFTR became stimulatable by CNP and was similarly inhibited by H-89, pointing to cross-activation of PKA. 8-pCPT-cGMP, a relatively cGKII-selective cGMP, failed to stimulate sCFTR. Another membrane-permeable and non-hydrolyzable analog of cGMP, 8-Br-cGMP, stimulated CFTR only at millimolar concentrations, consistent with cross-activation of PKA. The PDE inhibitors EHNA, rolipram, cilostamide, and amrinone did not significantly increase chloride conductance, arguing against a significant role for PDE2, PDE3 and PDE4 signaling in the oocyte. Sildenafil, a PDE5 inhibitor, caused a partial activation of sCFTR channels and this effect was again inhibited by H-89. CONCLUSION: From these experiments we conclude that in the Xenopus oocyte system, natriuretic peptides, 8-Br-cGMP, and PDE5 inhibitors activate CFTR by cross-activation of PKA.