Xenopus Oocyte Expression System Research Articles

Functional reconstitution of rat Nav1.6 sodium channels in vitro for studies of pyrethroid action

The ability to reconstitute sodium channel function and pharmacology in vitro using cloned subunits of known structure has greatly enhanced our understanding of the action of pyrethroid insecticides at this target and the structural determinants of resistance and interspecies selectivity. However, the use of reconstituted channels raises three critical questions: (1) Which subunits and subunit combinations should be used? (2) Which heterologous expression system is preferred? (3) Which combination of subunits and expression system best represents the function of native neuronal channels in the organism of interest? This review considers these questions from the perspective of recent research in this laboratory on the action of pyrethroid insecticides on rat Nav1.6 sodium channels by comparing the effects of heteroligomeric complex composition on channel function and insecticide response when channels are expressed in either Xenopus oocytes or stably-transformed HEK293 cells. These comparisons provide new insight into the influence of cellular context on the functional and pharmacological properties of expressed channels, the modulatory effects of sodium channel auxiliary subunits on the action of pyrethroids, and the relative fidelity of the Xenopus oocyte and HEK293 cell expression systems as model systems for studying of channel function and pyrethroid action.

A Protein Expression System for Evaluating Cation-PI And PI-PI Interaction in Proteins

In proteins phenylalanine, tyrosine, and tryptophan can be involved in cation-pi and pi-pi interactions. The importance of these interactions for protein structure and functioning is well recognized but measuring their strength experimentally is problematic. The chemical aminoacylation strategy combined with the Xenopus oocyte expression system was developed by the Dougherty group at Caltech for the production of proteins labeled with fluorinated aromatic amino acids involved in cation-pi interactions. This elegant expression system only yields ng quantities of proteins, limiting the scope to channel-forming proteins analyzed via patch-clamp. We developed a protein expression platform for the evaluation of pi-pi and cation-pi interactions involving tryptophan. This Lactococcus lactis based expression system can produce mg amounts of a set of 6 proteins, each carrying a different electron density in the tryptophan side chain. It was used to express variants of the multidrug repressor protein LmrR. LmrR regulates the drug-induced expression of the ABC type multidrug efflux transporter LmrCD. LmrR was crystallized in its drug-free state and in complex with aromatic substrates showing each substrate is sandwiched between two tryptophan residues, suggesting pi-pi interactions are operative. The importance of pi-pi interactions for LmrR to bind aromatic substrates could now be quantitated using fluorescence, surface plasmon resonance, and X-ray crystallography. In conclusion, the newly developed expression system makes possible a systematic approach to study cation-pi and pi-pi interactions in proteins using a wide range of biophysical methods.

Functional Expression and Characterization of Plant ABC Transporters in Xenopus laevis Oocytes for Transport Engineering Purposes.

Transport engineering in bioengineering is aimed at efficient export of the final product to reduce toxicity and feedback inhibition and to increase yield. The ATP-binding cassette (ABC) transporters with their highly diverse substrate specificity and role in cellular efflux are potentially suitable in transport engineering approaches, although their size and high number of introns make them notoriously difficult to clone. Here, we report a novel in planta "exon engineering" strategy for cloning of full-length coding sequence of ABC transporters followed by methods for biochemical characterization of ABC exporters in Xenopus oocytes. Although the Xenopus oocyte expression system is particularly suitable for expression of membrane proteins and powerful in screening for novel transporter activity, only few examples of successful expression of ABC transporter has been reported. This raises the question whether the oocytes system is suitable to express and characterize ABC transporters. Thus we have selected AtABCG25, previously characterized in insect cells as the exporter of commercially valuable abscisic acid-as case study for optimizing of characterization in Xenopus oocytes. The tools provided will hopefully contribute to more successful transport engineering in synthetic biology.

Activity of the enantiomers of erythro-3-hydroxyaspartate at glutamate transporters and NMDA receptors.

The enantiomers of erythro-3-hydroxyaspartate were tested for activity at glutamate transporters and NMDA receptors. Both enantiomers inhibited glutamate transporters in rat hippocampal crude synaptosomes and elicited substrate-like activity at excitatory amino acid transporter 1, 2, and 3 as measured by voltage clamp in the Xenopus oocyte expression system. The enantiomers had similar affinities, but the D-enantiomer showed a lower maximal effect at excitatory amino acid transporter 1, 2, and 3 than the L-enantiomer. Surprisingly, D-erythro-3-hydroxyaspartate was a potent NMDA receptor agonist with an EC50 value in rat hippocampal neurons of 320nM, whereas the L-enantiomer was 100-fold less potent. L-erythro-3-hydroxyaspartate showed activity at both glutamate transporters and NMDA receptors at concentrations that are reported to inhibit serine racemase, indicating a lack of selectivity. This enantiomeric pair may assist in shedding further light on the structural requirements for substrate activity at glutamate transporters and for agonist activity at NMDA receptors. The erythro enantiomers of 3-hydroxyaspartate had interesting and surprising effects on glutamate neurotransmitter systems. L-erythro-3-hydroxyaspartate had activity at both glutamate transporters (EAAT1/2/3) and NMDA receptors. D-erythro-3-hydroxyaspartate acted on EAATs, but was also identified as a highly potent NMDA receptor agonist. These enantiomers shed further light on the structural requirements for activity at EAATs and NMDA receptors.

The α4 isoform of the Na⁺,K⁺-ATPase is tuned for changing extracellular environments.

In their journey from the male to the female reproductive tract, spermatozoa are confronted with a constantly changing environment. To cope with the associated challenges, spermatozoa express a distinct set of transporters, channels and pumps. One of the membrane proteins unique to spermatozoa is the α4 isoform of the Na(+) ,K(+) -ATPase. In addition to α4, spermatozoa express the ubiquous α1 variant. To get a detailed understanding of how α1 and α4 differ, and why spermatozoa need an additional Na(+) ,K(+) -ATPase, we have conducted an electrophysiological comparison of the rodent isoforms (rat α4 versus mouse α1-3) using the Xenopus oocyte expression system. We demonstrate isoform-specific differences in the voltage sensitivity of steady-state turnover, with α2 being the more sensitive, and α1 and α2 having faster Na(+) release kinetics than α3 and α4. Our data further show that the α1 and α2 turnover rates are fast compared with those of α3 and α4. Finally, α4 is less influenced by changes in extracellular Na(+) and temperature than α1. Based on these findings, we discuss the possibility that evolution has selected robust activity rather than rapid turnover for α4.

Hereditary Renal Hypouricemia Type 1 and Autosomal Dominant Polycystic Kidney Disease

BackgroundRenal hypouricemia (RHUC) is a heterogeneous inherited disorder characterized by impaired tubular uric acid (UA) transport with severe complications, such as acute kidney injury. Type 1 is caused by a mutation in the SLC22A12 gene (URAT1) and type 2 in the SLC2A9 gene (GLUT9). In this article, the authors present a coexpression functional characterization of variants responsible for RHUC type 1 in a Czech family with polycystic kidney disease (PKD). MethodsThe serum UA concentration in the proband was 1.1 mg/dL and was expressed as an increase in the fractional excretion of UA (43%). The URAT1 allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. Linkage analysis using a panel of 4 CA-repeat flanking markers for chromosome 16 and a panel of 3 CA-repeat flanking markers for chromosome 4 was performed, which carry the PKD1 and PKD2 genes, respectively. ResultsCoexpression results showed that p.G366R and p.R477H suppressed urate transport by wild types. Colocalization studies showed an accumulation of URAT1 in the endoplasmic reticulum of the p.G366R variant and mainly retention of wild-type protein by variants p.G366R and p.R477H. ConclusionsThe findings suggest that not only loss-of-function mutation of URAT1 but also the dominant-negative effect cause RHUC through loss of UA absorption, partly due to protein misfolding caused by accumulation of URAT1 protein in the endoplasmic reticulum.

Pheromone binding proteins enhance the sensitivity of olfactory receptors to sex pheromones in Chilo suppressalis

Sexual communication in moths offers a simplified scenario to model and investigate insect sensory perception. Both PBPs (pheromone-binding proteins) and PRs (pheromone receptors) are involved in the detection of sex pheromones, but the interplay between them still remains largely unknown. In this study, we have measured the binding affinities of the four recombinant PBPs of Chilo suppressalis (CsupPBPs) to pheromone components and analogs and characterized the six PRs using the Xenopus oocytes expression system. Interestingly, when the responses of PRs were recorded in the presence of PBPs, we measured in several combinations a dramatic increase in signals as well as in sensitivity of such combined systems. Furthermore, the discrimination ability of appropriate combinations of PRs and PBPs was improved compared with the performance of PBPs or PRs alone. Besides further supporting a role of PBPs in the pheromone detection and discrimination, our data shows for the first time that appropriate combinations of PRs and PBPs improved the discrimination ability of PBPs or PRs alone. The variety of responses measured with different pairing of PBPs and PRs indicates the complexity of the olfaction system, which, even for the relatively simple task of detecting sex pheromones, utilises a highly sophisticated combinatorial approach.

Abstract 348: Amino acid-based prodrugs of gemcitabine - a therapeutic option to overcome chemoresistance in pancreatic cancer

Abstract Pancreatic cancer is the deadliest of all cancers. Currently, gemcitabine is the only drug of choice to treat this cancer but the results are modest as the disease itself is diagnosed at an advanced stage and often times develops resistance to gemcitabine via silencing of the influx transporters for the drug. We hypothesized that the delivery of gemcitabine into pancreatic cancer cells in a different form via some other transporters would provide a logical alternative approach. It would even be better if we can exploit for this purpose the transporters that are selectively upregulated in pancreatic cancer. Several nucleoside drugs are transportable substrates for the amino acid transporter SLC6A14 if used in the form of amino acid-based prodrugs. Such strategy would work for gemcitabine if SLC6A14 is upregulated in pancreatic cancer. With this in mind, we analyzed publically available microarray datasets for the amino acid transporters that are upregulated in pancreatic cancer. We found four transporters (SLC1A5, SLC7A5, SLC7A11, and SLC6A14) overexpressed in tumors compared to normal tissue. The fold-increase in expression is the highest for SLC6A14 in all datasets (range, 1.3 - 163.3; p, 0.05 - 2×10−14). The fold-increase for the other three transporters is 2 at the maximum. We confirmed these findings using primary pancreatic cancer tissues and cancer cell lines. First, we used pancreatic cancer tissues and compared with the normal tissues. SLC6A14 mRNA is increased at least by 7-fold in tumors versus normal tissue. We then evaluated the expression of SLC6A14 mRNA and protein in normal and pancreatic cancer cell lines. The expression is higher in all cancer cell lines than in a normal cell line. This was further confirmed with a tissue microarray containing pancreatic tumor tissues from 52 patients. The delivery of gemcitabine in the form of amino acid-based prodrugs via SLC6A14, an exceptionally energy-coupled transporter, as a means to overcome gemcitabine resistance is a novel idea that has never been tested. As a proof of principle, we have shown that SLC6A14 can transport several nucleoside drugs (acyclovir, ganciclovir, and cytarabine) in the form of amino acid-based prodrugs. The parent nucleosides are not substrates for the transporter. To specifically assess the interaction of amino acid-based prodrugs of gemcitabine with SLC6A14, we synthesized the valyl ester of gemcitabine and investigated its transport via SLC6A14 using two different heterologous expression systems: a mammalian cell expression system and the Xenopus oocyte expression system. In both experimental approaches, we found that the valyl ester of gemcitabine, but not the parent drug, is a transportable substrate for SLC6A14. The transport process is Na/Cl-coupled and electrogenic. We conclude that amino acid-based prodrugs of gemcitabine represent a viable alternative to overcome resistance to gemcitabine in the treatment of pancreatic cancer. Citation Format: Yangzom D. Bhutia, Ellappan Babu, Pankaj K. Singh, Vadivel Ganapathy. Amino acid-based prodrugs of gemcitabine - a therapeutic option to overcome chemoresistance in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 348. doi:10.1158/1538-7445.AM2015-348

Herbivory-induced glucose transporter gene expression in the brown planthopper, Nilaparvata lugens

Nilaparvata lugens, the brown planthopper (BPH) feeds on rice phloem sap, containing high amounts of sucrose as a carbon source. Nutrients such as sugars in the digestive tract are incorporated into the body cavity via transporters with substrate selectivity. Eighteen sugar transporter genes of BPH (Nlst) were reported and three transporters have been functionally characterized. However, individual characteristics of NlST members associated with sugar transport remain poorly understood. Comparative gene expression analyses using oligo-microarray and quantitative RT-PCR revealed that the sugar transporter gene Nlst16 was markedly up-regulated during BPH feeding. Expression of Nlst16 was induced 2 h after BPH feeding on rice plants. Nlst16, mainly expressed in the midgut, appears to be involved in carbohydrate incorporation from the gut cavity into the hemolymph. Nlst1 (NlHT1), the most highly expressed sugar transporter gene in the midgut was not up-regulated during BPH feeding. The biochemical function of NlST16 was shown as facilitative glucose transport along gradients. Glucose uptake activity by NlST16 was higher than that of NlST1 in the Xenopus oocyte expression system. At least two NlST members are responsible for glucose uptake in the BPH midgut, suggesting that the midgut of BPH is equipped with various types of transporters having diversified manner for sugar uptake.

Characterization of the honeybee AmNaV1 channel and tools to assess the toxicity of insecticides.

Pollination is important for both agriculture and biodiversity. For a significant number of plants, this process is highly, and sometimes exclusively, dependent on the pollination activity of honeybees. The large numbers of honeybee colony losses reported in recent years have been attributed to colony collapse disorder. Various hypotheses, including pesticide overuse, have been suggested to explain the disorder. Using the Xenopus oocytes expression system and two microelectrode voltage-clamp, we report the functional expression and the molecular, biophysical, and pharmacological characterization of the western honeybee’s sodium channel (Apis Mellifera NaV1). The NaV1 channel is the primary target for pyrethroid insecticides in insect pests. We further report that the honeybee’s channel is also sensitive to permethrin and fenvalerate, respectively type I and type II pyrethroid insecticides. Molecular docking of these insecticides revealed a binding site that is similar to sites previously identified in other insects. We describe in vitro and in silico tools that can be used to test chemical compounds. Our findings could be used to assess the risks that current and next generation pesticides pose to honeybee populations.

Control of the Water Transport Activity of Barley HvTIP3;1 Specifically Expressed in Seeds.

Tonoplast intrinsic proteins (TIPs) are involved in the transport and storage of water, and control intracellular osmotic pressure by transporting material related to the water potential of cells. In the present study, we focused on HvTIP3;1 during the periods of seed development and desiccation in barley. HvTIP3;1 was specifically expressed in seeds. An immunochemical analysis showed that HvTIP3;1 strongly accumulated in the aleurone layers and outer layers of barley seeds. The water transport activities of HvTIP3;1 and HvTIP1;2, which also accumulated in seeds, were measured in the heterologous expression system of Xenopus oocytes. When they were expressed individually, HvTIP1;2 transported water, whereas HvTIP3;1 did not. However, HvTIP3;1 exhibited water transport activity when co-expressed with HvTIP1;2 in oocytes, and this activity was higher than when HvTIP1;2 was expressed alone. This is the first report to demonstrate that the water permeability of a TIP aquaporin was activated when co-expressed with another TIP. The split-yellow fluorescent protein (YFP) system in onion cells revealed that HvTIP3;1 interacted with HvTIP1;2 to form a heterotetramer in plants. These results suggest that HvTIP3;1 functions as an active water channel to regulate water movement through tissues during the periods of seed development and desiccation.

A precise spacing between the NPA domains of aquaporins is essential for silicon permeability in plants.

The controversy surrounding silicon (Si) benefits and essentiality in plants is exacerbated by the differential ability of species to absorb this element. This property is seemingly enhanced in species carrying specific nodulin 26-like intrinsic proteins (NIPs), a subclass of aquaporins. In this work, our aim was to characterize plant aquaporins to define the features that confer Si permeability. Through comparative analysis of 985 aquaporins in 25 species with differing abilities to absorb Si, we were able to predict 30 Si transporters and discovered that Si absorption is exclusively confined to species that possess NIP-III aquaporins with a GSGR selectivity filter and a precise distance of 108 amino acids (AA) between the asparagine-proline-alanine (NPA) domains. The latter feature is of particular significance since it had never been reported to be essential for Si selectivity. Functionality assessed in the Xenopus oocyte expression system showed that NIPs with 108 AA spacing exhibited Si permeability, while proteins differing in that distance did not. In subsequent functional studies, a Si transporter from poplar mutated into variants with 109- or 107-AA spacing failed to import, and a tomato NIP gene mutated from 109 to 108 AA exhibited a rare gain of function. These results provide a precise molecular basis to classify higher plants into Si accumulators or excluders.

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.

The Effect of WNK4 on the Na+:Cl‐ Cotransporter is modulated by Intracellular Chloride

IntroductionThe effect of WNK4 on the renal Na+:Cl‐ cotransporter, NCC is controversial since several studies in vitro and in vivo have shown negative or positive effects. We hypothesize that both effects coexist and are modulated by the intracellular chloride concentration ([Cl‐]i). Piala et al. (2014) showed that L‐WNK1 is a Cl‐‐sensitive kinase and identified two leucine residues forming the Cl‐‐binding site. These leucines are conserved in WNK isoforms.Materials and MethodsWe used the functional expression system of Xenopus oocytes and HEK293 cells. NCC activity was measured by 22Na+ uptake and/or amino acid threonine phosphorylation in the absence or presence of wild type or mutant WNK4, in control conditions, or after [Cl‐]i depleting maneuvers. WNK4 autophosphorylation was also assessed in these conditions.ResultsAt baseline [Cl‐]i ∼ 45 mM, WNK4 autophosphorylation was undetectable and NCC activity was either inhibited or unaffected. [Cl‐]i reduction (25‐30 mM) promoted autophosphorylation transforming WNK4 to an activator of NCC in a kinase‐dependent manner. The L322F substitution transformed WNK4 into a constitutively autophosphorylated kinase that activated NCC without chloride depletion.ConclusionWNK4 is Cl‐ sensitive kinase exerting differential effects on NCC depending on the [Cl‐]i. It is possible that small changes in [Cl‐]i in distal convoluted tubule cells may have a crucial role determining WNK4 effect on NCC, suggesting that diverse physiological processes could converge in the Cl‐modulation of WNK's.

Transport of 4‐Boronophenylalanine, a BNCT Delivery Agent, by L‐Type Amino Acid Transporters 1 and 2

Boron neutron capture therapy (BNCT) is a biochemically-targeted radiotherapy based on the nuclear capture and fission reactions that occur when boron-10 is irradiated with low energy thermal neutrons to yield high linear energy transfer alpha particles and recoiling lithium-7 nuclei with path-length less than 10 μm.It has been focused to use on the treatment of brain, skin, head and neck cancers. 4-Boronophenylalanine (BPA), a BNCT delivery agent, has been used in clinical field but its transport mechanism into the tumor lesion was not clear. It is known that L-type amino acid transporters, LAT1 and LAT3 are highly expressed in various types of cancer whereas LAT2 and LAT4 are expressed in normal tissues. The LAT1-selective compound(s) might be the ideal delivery agents for BNCT to obtain the specific accumulation in cancer tissue and reduce adverse effect on normal tissue. In this study we identify the transport of BPA by L-type amino acid transporters using Xenopus oocyte expression system and LC/MS-MS analysis. Both L- and DL-BPA interacted well with LAT1 and LAT2, whereas the D-isoform showed lower interaction. All 3 isoforms of BPA did not interact with LAT3 but showed slightly interacted with LAT4 at concentration higher than 100 μM. Using LC-MS/MS analysis, L-and DL-BPA could be transported into cells by both LAT1 and LAT2. It means that BPA is not LAT1-selective substrate and might not be a suitable BNCT delivery agent. The ideal LAT1-selective substrates are need for further clinical trial. (This study is supported by the Grant-in-Aid from MEXT-supported Program for the Strategic Research Foundation at Private Universities)

Identification of rice cornichon as a possible cargo receptor for the Golgi-localized sodium transporter OsHKT1;3.

Membrane proteins are synthesized and folded in the endoplasmic reticulum (ER), and continue their path to their site of residence along the secretory pathway. The COPII system has been identified as a key player for selecting and directing the fate of membrane and secretory cargo proteins. Selection of cargo proteins within the COPII vesicles is achieved by cargo receptors. The cornichon cargo receptor belongs to a conserved protein family found in eukaryotes that has been demonstrated to participate in the selection of integral membrane proteins as cargo for their correct targeting. Here it is demonstrated at the cellular level that rice cornichon OsCNIH1 interacts with OsHKT1;3 and, in yeast cells, enables the expression of the sodium transporter to the Golgi apparatus. Physical and functional HKT-cornichon interactions are confirmed by the mating-based split ubiquitin system, bimolecular fluorescence complementation, and Xenopus oocyte and yeast expression systems. The interaction between the two proteins occurs in the ER of plant cells and their co-expression in oocytes leads to the sequestration of the transporter in the ER. In the yeast cornichon mutant erv14, OsHKT1;3 is mistargeted, preventing the toxic effects of sodium transport in the cell observed in wild-type cells or in the erv14 mutant that co-expressed OsHKT1;3 with either OsCNIH1 or Erv14p. Identification and characterization of rice cornichon as a possible cargo receptor opens up the opportunity to improve our knowledge on membrane protein targeting in plant cells.

NPT1/SLC17A1 is a renal urate exporter in humans and its common gain-of-function variant decreases the risk of renal underexcretion gout.

Serum uric acid (SUA) levels in humans are mainly regulated by urate transporters. Recent genome-wide association studies suggested that common variants of the human sodium-dependent phosphate cotransporter type 1 gene (NPT1/SLC17A1) influence SUA. NPT1 has been reported to mediate urate transport, but its physiologic role in regulating SUA in humans remains unclear. Furthermore, the findings of replication studies of the relationship between NPT1 variants and gout have been inconsistent. The aims of this study were to investigate the effect of NPT1 on gout and to determine its physiologic role. Five hundred forty-five male Japanese patients with gout and 1,115 male Japanese control subjects were genotyped for rs1165196 (I269T), a common missense variant in NPT1. Analyses of the association between rs1165196 and gout were then conducted, focusing especially on renal underexcretion (RUE) gout. Immunohistochemical analysis and functional analysis using Xenopus oocytes were also performed. Single-nucleotide polymorphism rs1165196 significantly decreased the risk of RUE gout (odds ratio 0.73, P = 0.031) but did not confer a risk for all gout (P = 0.123). The immunohistochemical analysis revealed that human NPT1 is localized to the apical membrane of the renal proximal tubule. The functional analysis using Xenopus oocyte expression systems showed that rs1165196 increases NPT1-mediated urate export. This study showed that NPT1 is a urate exporter located in the renal proximal tubule in humans, and that its common gain-of-function variant, rs1165196, causes RUE gout, a major subtype of gout. These findings enable us to deepen our understanding of the physiologic role of NPT1 as a renal urate exporter as well as its pathophysiologic role in gout.

Complex analysis of urate transporters SLC2A9, SLC22A12 and functional characterization of non-synonymous allelic variants of GLUT9 in the Czech population: no evidence of effect on hyperuricemia and gout.

ObjectiveUsing European descent Czech populations, we performed a study of SLC2A9 and SLC22A12 genes previously identified as being associated with serum uric acid concentrations and gout. This is the first study of the impact of non-synonymous allelic variants on the function of GLUT9 except for patients suffering from renal hypouricemia type 2.MethodsThe cohort consisted of 250 individuals (150 controls, 54 nonspecific hyperuricemics and 46 primary gout and/or hyperuricemia subjects). We analyzed 13 exons of SLC2A9 (GLUT9 variant 1 and GLUT9 variant 2) and 10 exons of SLC22A12 by PCR amplification and sequenced directly. Allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. The functional studies were analyzed using the non-parametric Wilcoxon and Kruskall-Wallis tests; the association study used the Fisher exact test and linear regression approach.ResultsWe identified a total of 52 sequence variants (12 unpublished). Eight non-synonymous allelic variants were found only in SLC2A9: rs6820230, rs2276961, rs144196049, rs112404957, rs73225891, rs16890979, rs3733591 and rs2280205. None of these variants showed any significant difference in the expression of GLUT9 and in urate transport. In the association study, eight variants showed a possible association with hyperuricemia. However, seven of these were in introns and the one exon located variant, rs7932775, did not show a statistically significant association with serum uric acid concentration.ConclusionOur results did not confirm any effect of SLC22A12 and SLC2A9 variants on serum uric acid concentration. Our complex approach using association analysis together with functional and immunohistochemical characterization of non-synonymous allelic variants did not show any influence on expression, subcellular localization and urate uptake of GLUT9.

Class III antiarrhythmic drug dronedarone inhibits cardiac inwardly rectifying Kir2.1 channels through binding at residue E224.

Dronedarone is a novel class III antiarrhythmic drug that is widely used in atrial fibrillation. It has been shown in native cardiomyocytes that dronedarone inhibits cardiac inwardly rectifying current IK1 at high concentrations, which may contribute both its antifibrillatory efficacy and its potential proarrhythmic side effects. However, the underlying mechanism has not been studied in further detail to date. In the mammalian heart, heterotetrameric assembly of Kir2.x channels is the molecular basis of IK1 current. Therefore, we studied the effects of dronedarone on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Dronedarone inhibited Kir2.1 currents but had no effect on Kir2.2 or Kir2.3 currents. Onset of block was slow but completely reversible upon washout. Blockade of Kir2.1 channels did not exhibit strong voltage dependence or frequency dependence. In a screening with different Kir2.1 mutants lacking specific binding sites within the cytoplasmic pore region, we found that residue E224 is essential for binding of dronedarone to Kir2.1 channels. In conclusion, direct block of Kir2.1 channel subunits by dronedarone through binding at E224 may underlie its inhibitory effects on cardiac IK1 current.

Functional characterization of sex pheromone receptors in the purple stem borer, Sesamia inferens (Walker).

The sex pheromone communication system in moths is highly species-specific and extremely sensitive, and pheromone receptors (PRs) are thought to be the most important factors in males. In the present study, three full-length cDNAs encoding PRs were characterized from Sesamia inferens antennae. These three PRs were all male-specific in expression, but their relative expression levels were very different; SinfOR29 was 17- to 23-fold higher than the other two PRs. Phylogenetic and motif pattern analyses showed that these three PRs were allocated to different PR subfamilies with different motif patterns. Functional analysis using the heterologous expression system of Xenopus oocytes demonstrated that SinfOR29 specifically and sensitively responded to the major pheromone component, Z11-16:OAc [concentration for 50% of maximal effect (EC50 ) = 3.431 × 10(-7) M], while SinfOR21 responded robustly to a minor pheromone component Z11-16:OH (EC50 = 1.087 × 10(-6) M). SinfOR27, however, displayed no response to any of the three pheromone components, but, interestingly, it was sensitive to a non-sex pheromone component Z9,E12-14:OAc (EC50 = 1.522 × 10(-6) M). Our results provide insight into the molecular mechanisms of specificity and sensitivity of the sex pheromone communication system in moths.