Functional and pharmacological characterization of human Na+-carnitine cotransporter hOCTN2

2000 ◽  
Vol 279 (3) ◽  
pp. F584-F591 ◽  
Author(s):  
Carsten A. Wagner ◽  
Ulrike Lükewille ◽  
Simone Kaltenbach ◽  
Ivano Moschen ◽  
Angelika Bröer ◽  
...  
Keyword(s):  
Organic Cation ◽  
Organic Cation Transporter ◽  
Carnitine Deficiency ◽  
Significant Inhibition ◽  
Xenopus Laevis Oocytes ◽  
Channel Blockers ◽  
Pharmacological Characterization ◽  
Transporter Family ◽  
Carnitine Transport ◽  
Induced Currents

l-Carnitine is essential for the translocation of acyl-carnitine into the mitochondria for β-oxidation of long-chain fatty acids. It is taken up into the cells by the recently cloned Na+-driven carnitine organic cation transporter OCTN2. Here we expressed hOCTN2 in Xenopus laevis oocytes and investigated with two-electrode voltage- clamp and flux measurements its functional and pharmacological properties as a Na+-carnitine cotransporter. l-carnitine transport was electrogenic. The l-carnitine-induced currents were voltage and Na+ dependent, with half-maximal currents at 0.3 ± 0.1 mM Na+ at −60 mV. Furthermore,l-carnitine-induced currents were pH dependent, decreasing with acidification. In contrast to other members of the organic cation transporter family, hOCTN2 functions as a Na+-coupled carnitine transporter. Carnitine transport was stereoselective, with an apparent Michaelis-Menten constant ( K m) of 4.8 ± 0.3 μM for l-carnitine and 98.3 ± 38.0 μM for d-carnitine. The substrate specificity of hOCTN2 differs from rOCT-1 and hOCT-2 as hOCTN2 showed only small currents with classic OCT substrates such as choline or tetraethylammonium; by contrast hOCTN2 mediated transport of betaine. hOCTN2 was inhibited by several drugs known to induce secondary carnitine deficiency. Most potent blockers were the antibiotic emetine and the ion channel blockers quinidine and verapamil. The apparent IC50 for emetine was 4.2 ± 1.2 μM. The anticonvulsant valproic acid did not induce a significant inhibition of carnitine transport, pointing to a different mode of action. In summary, hOCTN2 mediates electrogenic Na+-dependent stereoselective high-affinity transport ofl-carnitine and Na+. hOCTN2 displays transport properties distinct from other members of the OCT family and is directly inhibited by several substances known to induce systemic carnitine deficiency.

scholarly journals OCTN Cation Transporters in Health and Disease

2013 ◽  
Vol 18 (8) ◽  
pp. 851-867 ◽  
Author(s):  
Lorena Pochini ◽  
Mariafrancesca Scalise ◽  
Michele Galluccio ◽  
Cesare Indiveri
Keyword(s):  
Large Scale ◽  
Intact Cell ◽  
Organic Cation Transporter ◽  
Carnitine Deficiency ◽  
Transmembrane Segments ◽  
Pharmacological Targets ◽  
Carnitine Transport ◽  
Health And Disease ◽  
Inflammatory Bowel ◽  
Molecular Bases

The three members of the organic cation transporter novel subfamily are known to be involved in interactions with xenobiotic compounds. These proteins are characterized by 12 transmembrane segments connected by nine short loops and two large hydrophilic loops. It has been recently pointed out that acetylcholine is a physiological substrate of OCTN1. Its transport could be involved in nonneuronal cholinergic functions. OCTN2 maintains the carnitine homeostasis, resulting from intestinal absorption, distribution to tissues, and renal excretion/reabsorption. OCTN3, identified only in mouse, mediates also carnitine transport. OCTN1 and OCTN2 are associated with several pathologies, such as inflammatory bowel disease, primary carnitine deficiency, diabetes, neurological disorders, and cancer, thus representing useful pharmacological targets. The function and interaction with drugs of OCTNs have been studied in intact cell systems and in proteoliposomes. The latter experimental model enables reduced interference from other transporters or enzyme pathways. Using proteoliposomes, the molecular bases of toxicity of some drugs have recently been revealed. Therefore, proteoliposomes represent a promising experimental tool suitable for large-scale molecular screening of interactions of OCTNs with chemicals regarding human health.

Charge-to-substrate ratio during organic cation uptake by rat OCT2 is voltage dependent and altered by exchange of glutamate 448 with glutamine

2009 ◽  
Vol 296 (4) ◽  
pp. F709-F722 ◽  
Author(s):  
Bernhard M. Schmitt ◽  
Dmitry Gorbunov ◽  
Peter Schlachtbauer ◽  
Brigitte Egenberger ◽  
Valentin Gorboulev ◽  
...  
Keyword(s):  
Organic Cation ◽  
Three Dimensional ◽  
Organic Cation Transporter ◽  
Tubular Damage ◽  
Xenopus Laevis Oocytes ◽  
Lactose Permease ◽  
Cation Uptake ◽  
Ion Substitution ◽  
Charge Excess ◽  
Organic Cation Transporter 2

Uptake of substrate and electric charge was measured simultaneously in voltage-clamped Xenopus laevis oocytes expressing rat organic cation transporter 2 (rOCT2). At 0 mV, saturating substrate concentrations induced uptake of more positive elementary charges than monovalent organic cations, with charge-to-substrate ratios of 1.5 for guanidinium+, 3.5 for tetraethylammonium+, and 4.0 for 1-methyl-4-phenylpyridinium+. At negative holding potentials, the charge-to-substrate ratios decreased toward unity. At 0 mV, charge-to-substrate ratios higher than unity were observed at different extracellular pH and after replacement of extracellular Na+, K+, Ca2+, Mg2+, and/or Cl−. Charge-to-substrate ratios were not influenced by intracellular succinate2− or glutarate2−. The effects of membrane potential and ion substitution strongly suggest that the surplus of transported positive charge is not generated by passive ion permeabilities. Rather, we hypothetize that small cations are taken up together with organic cation substrates whereas the outward reorientation of the empty transporter is electroneutral. Nonselective cotransport of small cations was supported by the three-dimensional structures of rOCT2 in its inward-facing and outward-facing conformations, which we determined by homology modeling based on known corresponding structures of H+-lactose permease of E. coli, and by functional analysis of OCT mutants. In our model, the innermost cavity of the outward-open binding cleft is negatively charged by Glu448 and Asp475, whereas the inward-open innermost cavity is electroneutral, containing Asp379, Asp475, Lys215, and Arg440. Substitution of Glu448 by glutamine reduced the charge-to-TEA+ ratio at 0 mV to unity. The observed charge excess associated with organic cation uptake into depolarized cells may contribute to tubular damage in renal failure.

scholarly journals Molecular Cloning and Characterization of NKT, a Gene Product Related to the Organic Cation Transporter Family That Is Almost Exclusively Expressed in the Kidney

1997 ◽  
Vol 272 (10) ◽  
pp. 6471-6478 ◽  
Author(s):  
Carlos E. Lopez-Nieto ◽  
Guofeng You ◽  
Kevin T. Bush ◽  
Elvino J. G. Barros ◽  
Davio R. Beier ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Gene Product ◽  
Organic Cation ◽  
Organic Cation Transporter ◽  
Transporter Family

scholarly journals MOLECULAR AND FUNCTIONAL IDENTIFICATION OF NOVEL HUMAN ORGANIC CATION TRANSPORTER FAMILY, OCTN1 AND OCTN2

1998 ◽  
Vol 13 (supplement) ◽  
pp. 106-107
Author(s):  
Hikaru YABUUCHI ◽  
Rikiya OHASHI ◽  
Kazuki SAKAMOTO ◽  
Jun-ichi NEZU ◽  
Asuka OKU ◽  
...  
Keyword(s):  
Organic Cation ◽  
Organic Cation Transporter ◽  
Functional Identification ◽  
Transporter Family
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