Cl- as a bona fide signaling ion.

Cl- is the major extracellular (Cl-out) and intracellular (Cl-in) anion whose concentration is actively regulated by multiple transporters. These transporters generate Cl- gradients across the plasma membrane and between the cytoplasm and intracellular organelles. [Cl-]in changes rapidly in response to cell stimulation and influences many physiological functions, as well as cellular and systemic homeostasis. However, less appreciated is the signaling function of Cl-. Cl- interacts with multiple proteins to directly modify their activity. This review highlights the signaling function of Cl- and argues that Cl- is a bona fide signaling ion, a function deserving extensive exploration.

Different Pharmacological Properties of GLUT9a and GLUT9b: Potential Implications in Preeclampsia.

BACKGROUND/AIMS: Glucose transporter 9 (GLUT9/SLC2A9) is the major regulator of uric acid homeostasis in humans. Hyperuricemia due to impaired regulation by GLUT9 in pregnancy is closely associated with preeclampsia. While GLUT9 is expressed in two alternative splice variants, GLUT9a and GLUT9b, with different subcellular localizations, no functional differences of the two splice variants are known to date. The aim of this study was to investigate the function of both GLUT9 isoforms. METHODS: To characterize the different pharmacological properties of GLUT9a and GLUT9b electrophysiological studies of these isoforms and their modified variants, i.e. NmodGLUT9a and NmodGLUT9b, were performed using a Xenopus laevis oocytes model. Currents were measured by an electrode voltage clamp system. RESULTS: Functional experiments unveiled that uric acid transport mediated by GLUT9a but not GLUT9b is chloride-dependent: Replacing chloride by different anions resulted in a 3.43±0.63-fold increase of GLUT9a- but not GLUT9b-mediated currents. However, replacement by iodide resulted in a loss of current for GLUT9a but not GLUT9b. Iodide inhibits GLUT9a with an IC50 of 35.1±6.7µM. Modification of the N-terminal domain leads to a shift of the iodide IC50 to 1200±228µM. Using molecular docking studies, we identified two positively charged residues H23 and R31 in the N-terminal domain of hGLUT9a which can explain the observed functional differences. CONCLUSION: To the best of our knowledge, this is the first study showing that the N-terminal domain of hGLUT9a has a unique regulatory function and the potential to interact with small negatively charged ions like iodide. These findings may have significant implications in our understanding of hyperuricemia-associated diseases, specifically during pregnancy.

Mutation in the Monocarboxylate Transporter 12 Gene Affects Guanidinoacetate Excretion but Does Not Cause Glucosuria.

A heterozygous mutation (c.643C>A; p.Q215X) in the monocarboxylate transporter 12-encoding gene MCT12 (also known as SLC16A12) that mediates creatine transport was recently identified as the cause of a syndrome with juvenile cataracts, microcornea, and glucosuria in a single family. Whereas the MCT12 mutation cosegregated with the eye phenotype, poor correlation with the glucosuria phenotype did not support a pathogenic role of the mutation in the kidney. Here, we examined MCT12 in the kidney and found that it resides on basolateral membranes of proximal tubules. Patients with MCT12 mutation exhibited reduced plasma levels and increased fractional excretion of guanidinoacetate, but normal creatine levels, suggesting that MCT12 may function as a guanidinoacetate transporter in vivo However, functional studies in Xenopus oocytes revealed that MCT12 transports creatine but not its precursor, guanidinoacetate. Genetic analysis revealed a separate, undescribed heterozygous mutation (c.265G>A; p.A89T) in the sodium/glucose cotransporter 2-encoding gene SGLT2 (also known as SLC5A2) in the family that segregated with the renal glucosuria phenotype. When overexpressed in HEK293 cells, the mutant SGLT2 transporter did not efficiently translocate to the plasma membrane, and displayed greatly reduced transport activity. In summary, our data indicate that MCT12 functions as a basolateral exit pathway for creatine in the proximal tubule. Heterozygous mutation of MCT12 affects systemic levels and renal handling of guanidinoacetate, possibly through an indirect mechanism. Furthermore, our data reveal a digenic syndrome in the index family, with simultaneous MCT12 and SGLT2 mutation. Thus, glucosuria is not part of the MCT12 mutation syndrome.

A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities.

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

Hyperuricemia during Pregnancy Leads to a Preeclampsia-Like Phenotype in Mice.

Hyperuricemia is a common feature in pregnancies compromised by pre-eclampsia, a pregnancy disease characterized by hypertension and proteinuria. The role of uric acid in the pathogenesis of pre-eclampsia remains largely unclear. The aim of this study was to investigate the effect of elevated uric acid serum levels during pregnancy on maternal blood pressure and neonatal outcome using two different murine knockout models. Non-pregnant liver-specific GLUT9 knockout (LG9KO) mice showed elevated uric acid serum concentrations but no hypertensive blood pressure levels. During pregnancy, however, blood pressure levels of these animals increased in the second and third trimester, and circadian blood pressure dipping was severely altered when compared to non-pregnant LG9KO mice. The impact of hyperuricemia on fetal development was investigated using a systemic GLUT9 knockout (G9KO) mouse model. Fetal hyperuricemia caused distinctive renal tissue injuries and, subsequently an impaired neonatal growth pattern. These findings provide strong evidence that hyperuricemia plays a major role in the pathogenesis of hypertensive pregnancy disorders such as pre-eclampsia. These novel insights may enable the development of preventive and therapeutic strategies for hyperuricemia-related diseases.

Glucose Transporter 9 (GLUT9) Plays an Important Role in the Placental Uric Acid Transport System.

BACKGROUND: Hyperuricemia is a common laboratory finding in pregnant women compromised by preeclampsia. A growing body of evidence suggests that uric acid is involved in the pathogenesis of preeclampsia. Glucose transporter 9 (GLUT9) is a high-capacity uric acid transporter. The aim of this study was to investigate the placental uric acid transport system, and to identify the (sub-) cellular localization of GLUT9. METHODS: Specific antibodies against GLUT9a and GLUT9b isoforms were raised, and human villous (placental) tissue was immunohistochemically stained. A systemic GLUT9 knockout (G9KO) mouse model was used to assess the placental uric acid transport capacity by measurements of uric acid serum levels in the fetal and maternal circulation. RESULTS: GLUT9a and GLUT9b co-localized with the villous (apical) membrane, but not with the basal membrane, of the syncytiotrophoblast. Fetal and maternal uric acid serum levels were closely correlated. G9KO fetuses showed substantially higher uric acid serum concentrations than their mothers. CONCLUSIONS: These findings demonstrate that the placenta efficiently maintains uric acid homeostasis, and that GLUT9 plays a key role in the placental uric acid transport system, at least in this murine model. Further studies investigating the role of the placental uric acid transport system in preeclampsia are eagerly needed.

A residue close to α1 loop F disrupts modulation of GABAA receptors by benzodiazepines while their binding is maintained.

Benzodiazepines act at the major isoforms of GABA type A receptors where they potentiate the current evoked by the agonist GABA. The underlying mechanism of this potentiation is poorly understood, but hypothesized to be related to the mechanism that links agonist binding to channel opening in these ligand activated ion channels. The loop F of the α(1) and the ß(2) subunit have been implicated in channel gating, and loop F of the γ(2) subunit in the modulation by benzodiazepines. We have identified the conservative point mutation Y168F located N-terminally of loop F in the α(1) subunit that fails to affect agonist properties. Interestingly, it disrupts modulation by benzodiazepines, but leaves high affinity binding to the benzodiazepine binding site intact. Modulation by barbiturates and neurosteroids is also unaffected. Residue α(1) Y168 is not located either near the binding pockets for GABA, or for benzodiazepines, or close to the loop F of the γ(2) subunit. Our results support the fact, that broader regions of ligand gated receptors are conformationally affected by the binding of benzodiazepines. We infer that also broader regions could contribute to signaling from GABA agonist binding to channel opening.

Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects.

The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupienniussalei venom.

Use of concatamers to study GABAA receptor architecture and function: application to delta-subunit-containing receptors and possible pitfalls.

Many membrane proteins, including the GABA(A) [GABA (gamma-aminobutyric acid) type A] receptors, are oligomers often built from different subunits. As an example, the major adult isoform of the GABA(A) receptor is a pentamer built from three different subunits. Theoretically, co-expression of three subunits may result in many different receptor pentamers. Subunit concatenation allows us to pre-define the relative arrangement of the subunits. This method may thus be used to study receptor architecture, but also the nature of binding sites. Indeed, it made possible the discovery of a novel benzodiazepine site. We use here subunit concatenation to study delta-subunit-containing GABA(A) receptors. We provide evidence for the formation of different functional subunit arrangements in recombinant alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors. As with all valuable techniques, subunit concatenation has also some pitfalls. Most of these can be avoided by carefully titrating and minimizing the length of the linker sequences joining the two linked subunits and avoiding inclusion of the signal sequence of all but the N-terminal subunit of a multi-subunit construct. Maybe the most common error found in the literature is that low expression can be overcome by simply overloading the expression system with genetic information. As some concatenated constructs result by themselves in a low level of expression, this erroneous assembly leading to receptor function may be promoted by overloading the expression system and leads to wrong conclusions.

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs.

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.

Establishment of a novel microscale thermophoresis ligand-binding assay for characterization of SLC solute carriers using oligopeptide transporter PepT1 (SLC15 family) as a model system.

INTRODUCTION: Membrane proteins represent roughly one third of the human proteome and many of them serve as targets of therapeutic drugs. An exception is the SLC solute carrier superfamily with only a handful of approved drugs targeting SLCs. Indeed, for many of the SLCs, the natural transport substrates are still unknown. A major limitation for SLCs has been the difficulty to thoroughly characterize these multimembrane spanning proteins. The intrinsic properties of membrane proteins with alternative hydrophobic and hydrophilic domains lead to instability, making the purification tasks even more challenging compared to soluble proteins. This issue also holds true for conventional ligand-binding assays (LBAs) which usually require high-quality, pure and concentrated protein samples. Herein, we report a novel binding assay strategy to overcome these issues, taking advantage of a unique combination of yeast expression and microscale thermophoresis (MST). Following yeast overexpression of SLC15A1/PepT1 ortholog from moss Physcomitrella patens, PepTPp, which exhibits remarkable similarity to human PepT1, the approach was validated using dipeptide glycylsarcosine (Gly-Sar) and antiviral prodrug valacyclovir as test substrates. METHOD: The originality of our approach is based on the comparative analysis of solubilized total membrane preparations with or without expression of the SLC target of interest, using a yeast strain (S. cerevisiae), in which the corresponding endogenous SLC homolog is depleted. MST is a recently developed technique that takes advantage of the properties of biomolecules in solution to migrate along a temperature gradient. Importantly, this migration is affected by substrate binding. It is being monitored by fluorescence using labelled SLC molecules in the presence of different ligand concentrations. RESULTS: We herein report a novel MST/yeast-based method to characterize binding of ligands to SLCs without the need for a prior SLC-purification step. For validation purposes, we used a close eukaryotic homolog of the human H+-coupled oligopeptide transporter PepT1 (SLC15A1) that mediates uptake of di-tripeptides and peptide-like drugs as a test model. This approach allowed the successful confirmation of the binding of Gly-Sar at the mM range and revealed for the first time the KD of the antiviral prodrug valacyclovir to the PepT1 homolog at around 50 µM. DISCUSSION: This novel LBA approach is independent of protein purification. It is suitable for drug discovery as it is upscalable to high throughput compound screening. It works well for SLC transporters which are underrepresented targets due to their difficulties to study them. Moreover, this approach could make a significant contribution toward "deorphanization" of SLCs, revealing their transport substrates.

Placental glucose transporter (GLUT)-1 is down-regulated in preeclampsia.

INTRODUCTION: Transplacental fetal glucose supply is predominantly regulated by glucose transporter-1 (GLUT1). Altered expression and/or function of GLUT1 may affect the intrauterine environment, which could compromise fetal development and may contribute to fetal programming. To date it is unknown whether placental GLUT1 is affected by preeclampsia, which is often associated with intrauterine growth restriction (IUGR). We addressed the hypothesis that preeclampsia leads to decreased expression and function of placental GLUT1. METHODS: Placentae were obtained following normal pregnancy and from pregnancies affected by preeclampsia. Washed villous tissue fragments were used to prepare syncytial microvillous (MVM) and basal plasma membranes (BM) microvesicles. GLUT1 protein and mRNA expression was assessed by western blot analysis and qPCR using Fast SYBR Green. A radio-labeled glucose up-take assay using placenta-derived syncytial microvesicles was used to analyze GLUT1 function. RESULTS: GLUT1 protein expression was significantly down-regulated in (apical) MVM of the syncytiotrophoblast in preeclampsia (n = 6) compared to controls (n = 6) (0.40 ± 0.04 versus 1.00 ± 0.06, arbitrary units, P < 0.001, Student's t-test), while GLUT1 mRNA expression did not show a significant difference. In addition, the functional assay in syncytial microvesicles showed a significantly decreased glucose transport activity in preeclampsia (61.78 ± 6.48%, P < 0.05) compared to controls. BM GLUT1 protein expression was unchanged and glucose up-take into BM microvesicles showed no differences between the preeclampsia and control groups. DISCUSSION: Our study shows for the first time that in preeclampsia placental GLUT1 expression and function are down-regulated at the apical plasma membrane of the syncytiotrophoblast. Further studies are needed to assess whether these changes occur also in vivo and contribute to the development of IUGR in preeclampsia.

Loss of Sodium/Hydrogen Exchanger NHA2 Exacerbates Obesity- and Aging-Induced Glucose Intolerance in Mice.

We previously demonstrated that the sodium/hydrogen exchanger NHA2, also known as NHEDC2 or SLC9B2, is critical for insulin secretion by ß-cells. To gain more insights into the role of NHA2 on systemic glucose homeostasis, we studied the impact of loss of NHA2 during the physiological aging process and in the setting of diet-induced obesity. While glucose tolerance was normal at 2 months of age, NHA2 KO mice displayed a significant glucose intolerance at 5 and 12 months of age, respectively. An obesogenic high fat diet further exacerbated the glucose intolerance of NHA2 KO mice. Insulin levels remained similar in NHA2 KO and WT mice during aging and high fat diet, but fasting insulin/glucose ratios were significantly lower in NHA2 KO mice. Peripheral insulin sensitivity, measured by insulin tolerance tests and hyperinsulinemic euglycemic clamps, was unaffected by loss of NHA2 during aging and high fat diet. High fat diet diminished insulin secretion capacity in both WT and NHA2 KO islets and reduced expression of NHA2 in WT islets. In contrast, aging was characterized by a gradual increase of NHA2 expression in islets, paralleled by an increasing difference in insulin secretion between WT and NHA2 KO islets. In summary, our results demonstrate that loss of the sodium/hydrogen exchanger NHA2 exacerbates obesity- and aging-induced glucose intolerance in mice. Furthermore, our data reveal a close link between NHA2 expression and insulin secretion capacity in islets.

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.

Influence of GABA(A) receptor α subunit isoforms on the benzodiazepine binding site.

Classical benzodiazepines, such as diazepam, interact with α(x)ß(2)γ(2) GABA(A) receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α(1)S204Cß(2)γ(2), α(1)S205Cß(2)γ(2) and α(1)T206Cß(2)γ(2) and with receptors containing the homologous mutations in α(2)ß(2)γ(2), α(3)ß(2)γ(2), α(5)ß(1/2)γ(2) and α(6)ß(2)γ(2). The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α(1)S204Cß(2)γ(2), α(1)S205Cß(2)γ(2) and α(1)T206Cß(2)γ(2) and homologous positions in α(2), α(3), α(5) and α(6) with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABA(A) receptor isoform α(1)ß(2)γ(2), α(2)ß(2)γ(2), α(3)ß(2)γ(2), α(5)ß(1/2)γ(2) and α(6)ß(2)γ(2).

A closer look at the high affinity benzodiazepine binding site on GABAA receptors.

Ligands of the benzodiazepine binding site of the GABA(A) receptor come in three flavors: positive allosteric modulators, negative allosteric modulators and antagonists all of which can bind with high affinity. The GABA(A) receptor is a pentameric protein which forms a chloride selective ion channel and ligands of the benzodiazepine binding site stabilize three different conformations of this protein. Classical benzodiazepines exert a positive allosteric effect by increasing the apparent affinity of channel opening by the agonist γ-aminobutyric acid (GABA). We concentrate here on the major adult isoform, the α(1)ß(2)γ(2) GABA(A) receptor. The classical binding pocket for benzodiazepines is located in a subunit cleft between α(1) and γ(2) subunits in a position homologous to the agonist binding site for GABA that is located between ß(2) and α(1) subunits. We review here approaches to this picture. In particular, point mutations were performed in combination with subsequent analysis of the expressed mutant proteins using either electrophysiological techniques or radioactive ligand binding assays. The predictive power of these methods is assessed by comparing the results with the predictions that can be made on the basis of the recently published crystal structure of the acetylcholine binding protein that shows homology to the N-terminal, extracellular domain of the GABA(A) receptor. In addition, we review an approach to the question of how the benzodiazepine ligands are positioned in their binding pocket. We also discuss a newly postulated modulatory site for benzodiazepines at the α(1)/ß(2) subunit interface, homologous to the classical benzodiazepine binding pocket.