scholarly journals Multifunctional ion transport properties of human SLC4A11: comparison of the SLC4A11-B and SLC4A11-C variants

2016 ◽  
Vol 311 (5) ◽  
pp. C820-C830 ◽  
Author(s):  
Liyo Kao ◽  
Rustam Azimov ◽  
Xuesi M. Shao ◽  
Ricardo F. Frausto ◽  
Natalia Abuladze ◽  
...  
Keyword(s):  
Ion Transport ◽  
Transport Properties ◽  
Late Onset ◽  
Sensorineural Deafness ◽  
Corneal Dystrophy ◽  
Hek 293 ◽  
Coupled Mode ◽  
293 Cells ◽  
Inward Currents ◽  
Dystrophic Disease

Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH2-terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H+ equivalents in both a Na+-independent and Na+-coupled mode. In both transport modes, SLC4A11-C H+ flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH2-terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH2-terminus functions as an autoactivating domain, enhancing Na+-independent and Na+-coupled H+ flux without significantly affecting the electrogenic NH3-H( n)+ cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11.

Functional characterization of testis-specific rodent multidrug and toxic compound extrusion 2, a class III MATE-type polyspecific H+/organic cation exporter

2007 ◽  
Vol 293 (5) ◽  
pp. C1437-C1444 ◽  
Author(s):  
Miki Hiasa ◽  
Takuya Matsumoto ◽  
Toshinori Komatsu ◽  
Hiroshi Omote ◽  
Yoshinori Moriyama
Keyword(s):  
Transport Properties ◽  
Leydig Cells ◽  
Functional Characterization ◽  
Organic Cations ◽  
Class Iii ◽  
Hek 293 ◽  
Toxic Compound ◽  
293 Cells ◽  
Carbonyl Cyanide ◽  
Liver And Kidney

Mammalian multidrug and toxic compound extrusion (MATE) proteins are classified into three subfamilies: classes I, II, and III. We previously showed that two of these families act as polyspecific H+-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney (Otsuka et al. Proc Natl Acad Sci USA 102: 17923–17928, 2005; Omote et al. Trends Pharmacol Sci 27: 587–593, 2006). Rodent MATE2 proteins are class III MATE transporters, the molecular nature, as well as transport properties, of which remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). On expression in human embryonic kidney (HEK)-293 cells, mMATE2 localized to the intracellular organelles and plasma membrane. mMATE2 mediated pH-dependent TEA transport with substrate specificity similar to, but distinct from, that of mMATE1, which prefers N-methylnicotinamide and guanidine as substrates. mMATE2 expressed in insect cells was solubilized and reconstituted with bacterial H+-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone but unaffected by valinomycin in the presence of K+. Immunologic techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus mMATE2 appears to act as a polyspecific H+/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporters of organic cations.

Inhibition of Na+-K+-2Cl−cotransport by mercury

1999 ◽  
Vol 277 (4) ◽  
pp. C684-C692 ◽  
Author(s):  
Steven C. Jacoby ◽  
Edith Gagnon ◽  
Luc Caron ◽  
John Chang ◽  
Paul Isenring
Keyword(s):  
Ion Transport ◽  
Binding Sites ◽  
Essential Role ◽  
Species Differences ◽  
Transmembrane Domain ◽  
Transport Proteins ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH−) groups. The inorganic form (Hg2+) is toxic to epithelial tissues and interacts with various transport proteins including the Na+ pump and Cl− channels. In this study, we determined whether the Na+-K+-Cl−cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg2+ on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg2+inhibits Na+-K+-Cl−cotransport, with inhibitor constant ( K i) values of 25 μM for the shark carrier (sNKCC1) and 43 μM for the human carrier. In further studies, we took advantage of species differences in Hg2+ affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697→Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+binding. We also show that modification of additional SH− groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.

Activation of Vanilloid Receptor 1 (VR1) by Eugenol

2003 ◽  
Vol 82 (10) ◽  
pp. 781-785 ◽  
Author(s):  
B.H. Yang ◽  
Z.G. Piao ◽  
Y.-B. Kim ◽  
C.-H. Lee ◽  
J.K. Lee ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Expression System ◽  
Sensory Nerve ◽  
Ruthenium Red ◽  
Vanilloid Receptor ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Vanilloid Receptor 1 ◽  
293 Cells ◽  
Inward Currents

The structural similarity of eugenol with capsaicin suggests that these two agents may share molecular mechanisms to produce their effects. We investigated the effects of eugenol in comparison with those of capsaicin using whole-cell patch clamp and Fura-2-based calcium-imaging techniques in a heterologous expression system and with sensory neurons. In vanilloid receptor 1 (VR1)-expressing human embryonic kidney (HEK) 293 cells and trigeminal ganglion (TG) neurons, eugenol activated inward currents, whereas capsazepine, a competitive VR antagonist, and ruthenium red (RR), a functional VR antagonist, completely blocked eugenol-induced inward currents. Moreover, eugenol caused elevation of [Ca2+]i, and this was completely abolished by both capsazepine and ruthenium red in VR1-expressing HEK 293 cells and TG neurons. Our results provide strong evidence that eugenol produces its effects, at least in part, via VR1 expressed by the sensory nerve endings in the teeth.

Wide variety of locations for rodent MATE1, a transporter protein that mediates the final excretion step for toxic organic cations

2006 ◽  
Vol 291 (4) ◽  
pp. C678-C686 ◽  
Author(s):  
Miki Hiasa ◽  
Takuya Matsumoto ◽  
Toshinori Komatsu ◽  
Yoshinori Moriyama
Keyword(s):  
Transport Properties ◽  
Leydig Cells ◽  
Wide Distribution ◽  
The Body ◽  
Organic Cations ◽  
Bladder Epithelium ◽  
Ito Cells ◽  
Hek 293 ◽  
Transporter Protein ◽  
293 Cells

MATE1 was the first mammalian example of the multidrug and toxin extrusion (MATE) protein family to be identified. Human MATE1 (hMATE1) is predominantly expressed and localized to the luminal membranes of the urinary tubules and bile canaliculi and mediates H+-coupled electroneutral excretion of toxic organic cations (OCs) into urine and bile (Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, and Moriyama Y. Proc Natl Acad Sci USA 102: 17923–17928, 2005). mMATE1, a mouse MATE ortholog, is also predominantly expressed in kidney and liver, although its transport properties are not yet characterized. In the present study, we investigated the transport properties and localization of mMATE1. Upon expression of this protein in HEK-293 cells, mMATE1 mediated electroneutral H+/tetraethylammonium exchange and showed a substrate specificity similar to that of hMATE1. Immunological techniques with specific antibodies against mMATE1 combined with RT-PCR revealed that mMATE1 is also expressed in various cells, including brain glia-like cells and capillaries, pancreatic duct cells, urinary bladder epithelium, adrenal gland cortex, α cells of the islets of Langerhans, Leydig cells, and vitamin A-storing Ito cells. These results indicate that mMATE1 is a polyspecific H+/OC exchanger. The unexpectedly wide distribution of mMATE1 suggests involvement of this transporter protein in diverse biological functions other than excretion of OCs from the body.

scholarly journals Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding

1998 ◽  
Vol 112 (5) ◽  
pp. 549-558 ◽  
Author(s):  
Paul Isenring ◽  
Steven C. Jacoby ◽  
John Chang ◽  
Bliss Forbush
Keyword(s):  
Ion Transport ◽  
Point Mutations ◽  
Cation Transport ◽  
Anion Transport ◽  
Binding Kinetics ◽  
Kinetic Behavior ◽  
Inhibitor Binding ◽  
Hek 293 ◽  
293 Cells ◽  
86Rb Influx

The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive 86Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human–shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2–7 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the Ki(bumetanide) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 Kis.

scholarly journals Permeation and Gating in CaV3.1 (α1G) T-type Calcium Channels Effects of Ca2+, Ba2+, Mg2+, and Na+

2008 ◽  
Vol 132 (2) ◽  
pp. 223-238 ◽  
Author(s):  
Nilofar Khan ◽  
I. Patrick Gray ◽  
Carlos A. Obejero-Paz ◽  
Stephen W. Jones
Keyword(s):  
Calcium Channels ◽  
Surface Charge ◽  
Concentration Dependence ◽  
Divalent Cations ◽  
Hek 293 ◽  
Voltage Range ◽  
293 Cells ◽  
Instantaneous Current ◽  
Voltage Dependent ◽  
Inward Currents

We examined the concentration dependence of currents through CaV3.1 T-type calcium channels, varying Ca2+ and Ba2+ over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current–voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by PCa/PNa = 87 and PCa/PBa = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent Kd values were similar for Ca2+ and Ba2+, both for block of currents carried by Na+ (3 μM for Ca2+ vs. 4 μM for Ba2+, at −30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca2+ vs. 2.5 mM for Ba2+; nearly voltage independent). Block by 3–10 μM Ca2+ was time dependent, described by bimolecular kinetics with binding at ∼3 × 108 M−1s−1 and voltage-dependent exit. Ca2+o, Ba2+o, and Mg2+o also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e− per 98 Å2 from Gouy-Chapman theory. Additionally, inward currents inactivated ∼35% faster in Ba2+o (vs. Ca2+o or Na+o). The accelerated inactivation in Ba2+o correlated with the transition from Na+ to Ba2+ permeation, suggesting that Ba2+o speeds inactivation by occupying the pore. We conclude that the selectivity of the “surface charge” among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca2+ or Ba2+.

A novel missense TGFBI variant p.(Ser591Phe) in a Finnish family with variant lattice corneal dystrophy

2021 ◽  
pp. 112067212199730
Author(s):  
Aino Maaria Jaakkola ◽  
Petri J Järventausta ◽  
Reetta-Stiina Järvinen ◽  
Pauliina Repo ◽  
Tero T Kivelä ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Late Onset ◽  
Family Members ◽  
Anterior Segment ◽  
Corneal Dystrophy ◽  
Ophthalmological Examination ◽  
Lattice Corneal Dystrophy ◽  
Tgfbi Gene ◽  
Novel Variant

Introduction: We describe the phenotype of a variant lattice corneal dystrophy (LCD) potentially caused by a novel variant c.1772C>T p.(Ser591Phe) in exon 13 of the transforming growth factor beta-induced (TGFBI) gene. Case report: The proband, a 71-year-old woman referred because of bilateral LCD, first seen at the age of 65 years, with recent progressive symptoms, underwent a clinical ophthalmological examination, anterior segment optical coherence tomography and confocal microscopy. Additionally, three siblings and three children were examined. The identified TGFBI variant was screened in six family members using Sanger sequencing. A corneal dystrophy gene screen was performed for the proband. Translucent subepithelial irregularities and central to midperipheral stubby branching corneal stromal lattice lines, asymmetric between the right and the left eye, were visible and resulted in mild deterioration of vision in one eye. Genetic testing revealed a novel variant c.1772C>T in TGFBI, leading to the amino acid change p.(Ser591Phe). One daughter carried the same variant but had only thick stromal nerve fibres at the age of 49 years. The other family members neither had corneal abnormalities nor carried the variant. No keratoplasty is yet planned for the proband. Conclusions: We classify the novel variant in TGFBI as possibly pathogenic, potentially causing the late-onset, asymmetric variant LCD. Our findings add to the growing number of TGFBI variants associated with a spectrum of phenotypes of variant LCD.

scholarly journals Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

2021 ◽  
Vol 22 (9) ◽  
pp. 4637
Author(s):  
Daniel Barth ◽  
Andreas Lückhoff ◽  
Frank J. P. Kühn
Keyword(s):  
Amino Acid Residues ◽  
Hek 293 ◽  
Complete Inactivation ◽  
293 Cells ◽  
Activation Mechanisms ◽  
Specific Regulation ◽  
Species Specific ◽  
Inside Out ◽  
Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

scholarly journals Preliminary Study on Pasta Samples Characterized in Antioxidant Compounds and Their Biological Activity on Kidney Cells

Nutrients ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 1131 ◽  
Author(s):  
Federico Di Marco ◽  
Francesco Trevisani ◽  
Pamela Vignolini ◽  
Silvia Urciuoli ◽  
Andrea Salonia ◽  
...  
Keyword(s):  
Indoleacetic Acid ◽  
Antioxidant Properties ◽  
Antioxidant Compounds ◽  
Kidney Cells ◽  
Hek 293 ◽  
293 Cells ◽  
High Concentrations ◽  
Β Carotene ◽  
Egg Pasta

Pasta is one of the basic foods of the Mediterranean diet and for this reason it was chosen for this study to evaluate its antioxidant properties. Three types of pasta were selected: buckwheat, rye and egg pasta. Qualitative–quantitative characterization analyses were carried out by HPLC-DAD to identify antioxidant compounds. The data showed the presence of carotenoids such as lutein and polyphenols such as indoleacetic acid, (carotenoids from 0.08 to 0.16 mg/100 g, polyphenols from 3.7 to 7.4 mg/100 g). To assess the effect of the detected metabolites, in vitro experimentation was carried out on kidney cells models: HEK-293 and MDCK. Standards of β-carotene, indoleacetic acid and caffeic acid, hydroalcoholic and carotenoid-enriched extracts from samples of pasta were tested in presence of antioxidant agent to determine viability variations. β-carotene and indoleacetic acid standards exerted a protective effect on HEK-293 cells while no effect was detected on MDCK. The concentrations tested are likely in the range of those reached in body after the consumption of a standard pasta meal. Carotenoid-enriched extracts and hydroalcoholic extracts showed different effects, observing rescues for rye pasta hydroalcoholic extract and buckwheat pasta carotenoid-enriched extract, while egg pasta showed milder dose depending effects assuming pro-oxidant behavior at high concentrations. The preliminary results suggest behaviors to be traced back to the whole phytocomplexes respect to single molecules and need further investigations.

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