Characterization of regulatory mechanisms and states of human organic cation transporter 2

2006 ◽  
Vol 290 (6) ◽  
pp. C1521-C1531 ◽  
Author(s):  
Jürgen Biermann ◽  
Detlef Lang ◽  
Valentin Gorboulev ◽  
Hermann Koepsell ◽  
Aleksandra Sindic ◽  
...  
Keyword(s):  
Organic Cation ◽  
Organic Cation Transporter ◽  
Binding Pocket ◽  
Organic Cations ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Organic Cation Transporter 2 ◽  
Interaction Domains ◽  
Specific Uptake

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]- N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 μM amiloride and 24 μM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in μM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (−55 ± 5%, n = 10 and −63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (−54 ± 5%, n = 14, and −31 ± 6%, n = 26) and PKA (−16 ± 5%, n = 16, and −18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (−22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking.

Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases

2003 ◽  
Vol 284 (2) ◽  
pp. F293-F302 ◽  
Author(s):  
Ibrahim Çetinkaya ◽  
Giuliano Ciarimboli ◽  
Gülay Yalçinkaya ◽  
Thomas Mehrens ◽  
Ana Velic ◽  
...  
Keyword(s):  
Myosin Light Chain ◽  
Organic Cation ◽  
Organic Cation Transporter ◽  
Substrate Affinity ◽  
Phosphatidylinositol 3 Kinase ◽  
Hek 293 ◽  
293 Cells ◽  
Dependent Signaling Pathway ◽  
Initial Uptake

Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]- N-methylpyridinium (ASP+). ASP+uptake was electrogenic and inhibited by TPA+(EC50= 2.7 μM), tetraethylammonium (EC50= 35 μM), cimetidine (EC50= 36 μM), or quinine (EC50= 6.7 μM). Stimulation with carbachol or ATP decreased initial uptake by 44 ± 3 ( n = 14) and 34 ± 4% ( n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 ± 4% ( n = 40). Inhibition of calmodulin (CaM), Ca2+/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 ± 2 ( n = 15), 40 ± 4 ( n = 30), and 31 ± 4% ( n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC50value for the inhibition of ASP+uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.

scholarly journals Properties of Transport Mediated by the Human Organic Cation Transporter 2 Studied in a Polarized Three-Dimensional Epithelial Cell Culture Model

2021 ◽  
Vol 22 (17) ◽  
pp. 9658
Author(s):  
Tim N. Koepp ◽  
Alexander Tokaj ◽  
Pavel I. Nedvetsky ◽  
Ana Carolina Conchon Costa ◽  
Beatrice Snieder ◽  
...  
Keyword(s):  
Organic Cation ◽  
Basolateral Membrane ◽  
Three Dimensional ◽  
Organic Cation Transporter ◽  
Organic Cations ◽  
Membrane Domain ◽  
Cell Culture Model ◽  
Hek Cells ◽  
Culture Model ◽  
Organic Cation Transporter 2

The renal secretory clearance for organic cations (neurotransmitters, metabolism products and drugs) is mediated by transporters specifically expressed in the basolateral and apical plasma membrane domains of proximal tubule cells. Here, human organic cation transporter 2 (hOCT2) is the main transporter for organic cations in the basolateral membrane domain. In this study, we stably expressed hOCT2 in Madin-Darby Canine Kidney (MDCK) cells and cultivated these cells in the presence of an extracellular matrix to obtain three-dimensional (3D) structures (cysts). The transport properties of hOCT2 expressed in MDCK cysts were compared with those measured using human embryonic kidney cells (HEK293) stably transfected with hOCT2 (hOCT2-HEK cells). In the MDCK cysts, hOCT2 was expressed in the basolateral membrane domain and showed a significant uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) with an affinity (Km) of 3.6 ± 1.2 µM, similar to what was measured in the hOCT2-HEK cells (Km = 3.1 ± 0.2 µM). ASP+ uptake was inhibited by tetraethylammonium (TEA+), tetrapentylammonium (TPA+), metformin and baricitinib both in the hOCT2-HEK cells and the hOCT2- MDCK cysts, even though the apparent affinities of TEA+ and baricitinib were dependent on the expression system. Then, hOCT2 was subjected to the same rapid regulation by inhibition of p56lck tyrosine kinase or calmodulin in the hOCT2-HEK cells and hOCT2- MDCK cysts. However, inhibition of casein kinase II regulated only activity of hOCT2 expressed in MDCK cysts and not in HEK cells. Taken together, these results suggest that the 3D cell culture model is a suitable tool for the functional analysis of hOCT2 transport properties, depending on cell polarization.

Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter

2008 ◽  
Vol 294 (4) ◽  
pp. C1074-C1078 ◽  
Author(s):  
Takuya Matsumoto ◽  
Takuji Kanamoto ◽  
Masato Otsuka ◽  
Hiroshi Omote ◽  
Yoshinori Moriyama
Keyword(s):  
Organic Cation ◽  
Ph Dependence ◽  
Substrate Recognition ◽  
Transport Activity ◽  
Hek 293 ◽  
Mutant Proteins ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Transmembrane Regions

Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H+/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in Vmax value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.

scholarly journals Molecular and cellular physiology of organic cation transporter 2

2019 ◽  
Vol 317 (6) ◽  
pp. F1669-F1679 ◽  
Author(s):  
Stephen H. Wright
Keyword(s):  
Organic Cation ◽  
Critical Role ◽  
Organic Cation Transporter ◽  
Initial Step ◽  
Organic Cations ◽  
Molecular Characteristics ◽  
Ligand Interaction ◽  
Organic Cation Transporter 2 ◽  
Influence Of Substrate ◽  
Inhibitory Potential

Organic cation transporters play a critical role in mediating the distribution of cationic pharmaceuticals. Indeed, organic cation transporter (OCT)2 is the initial step in the renal secretion of organic cations and consequently plays a defining role in establishing the pharmacokinetics of many cationic drugs. Although a hallmark of OCTs is their broad selectivity, this characteristic also makes them targets for unwanted, adverse drug-drug interactions (DDIs), making them a focus for efforts to develop models of ligand interaction that could predict and preempt these adverse interactions. This review discusses the molecular characteristics of these transporters as well as the evidence that established the OCTs as key players in the distribution of organic cations. However, the primary focus is the present understanding of the complexity of ligand interaction with OCTs, particularly OCT2, including evidence for the presence of multiple ligand-binding sites and the influence of substrate structure on the affinity of the transporter for inhibitory ligands. This leads to a discussion of the complexities associated with the development of protocols for assessing the inhibitory potential of new molecular entities to perpetrate unwanted DDIs, the criteria that should be considered in the interpretation of the results of such protocols, and the challenges associated with development of models capable of predicting unwanted DDIs.

scholarly journals Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽  
2013 ◽  
Vol 9 (9) ◽  
pp. 1407-1417 ◽  
Author(s):  
Patience Musiwaro ◽  
Matthew Smith ◽  
Maria Manifava ◽  
Simon A. Walker ◽  
Nicholas T. Ktistakis
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

2005 ◽  
Vol 103 (6) ◽  
pp. 1156-1166 ◽  
Author(s):  
Kevin J. Gingrich ◽  
Son Tran ◽  
Igor M. Nikonorov ◽  
Thomas J. Blanck
Keyword(s):  
Charge Transfer ◽  
Calcium Channels ◽  
Dependent Manner ◽  
Current Time ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

2007 ◽  
Vol 9 (4) ◽  
pp. 475-485 ◽  
Author(s):  
R. M. Johann ◽  
Ch. Baiotto ◽  
Ph. Renaud
Keyword(s):  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells
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