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Nicola Longo - One of the best experts on this subject based on the ideXlab platform.
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Carnitine transport and fatty acid oxidation
Biochimica et Biophysica Acta, 2016Co-Authors: Nicola Longo, Marta Frigeni, Marzia PasqualiAbstract:Carnitine is essential for the transfer of long-chain fatty acids across the inner mitochondrial membrane for subsequent β-oxidation. It can be synthesized by the body or assumed with the diet from meat and dairy products. Defects in Carnitine biosynthesis do not routinely result in low plasma Carnitine levels. Carnitine is accumulated by the cells and retained by kidneys using OCTN2, a high affinity organic cation transporter specific for Carnitine. Defects in the OCTN2 Carnitine transporter results in autosomal recessive primary Carnitine Deficiency characterized by decreased intracellular Carnitine accumulation, increased losses of Carnitine in the urine, and low serum Carnitine levels. Patients can present early in life with hypoketotic hypoglycemia and hepatic encephalopathy, or later in life with skeletal and cardiac myopathy or sudden death from cardiac arrhythmia, usually triggered by fasting or catabolic state. This disease responds to oral Carnitine that, in pharmacological doses, enters cells using the amino acid transporter B(0,+). Primary Carnitine Deficiency can be suspected from the clinical presentation or identified by low levels of free Carnitine (C0) in the newborn screening. Some adult patients have been diagnosed following the birth of an unaffected child with very low Carnitine levels in the newborn screening. The diagnosis is confirmed by measuring low Carnitine uptake in the patients' fibroblasts or by DNA sequencing of the SLC22A5 gene encoding the OCTN2 Carnitine transporter. Some mutations are specific for certain ethnic backgrounds, but the majority are private and identified only in individual families. Although the genotype usually does not correlate with metabolic or cardiac involvement in primary Carnitine Deficiency, patients presenting as adults tend to have at least one missense mutation retaining residual activity. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.
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glycosylation of the octn2 Carnitine transporter study of natural mutations identified in patients with primary Carnitine Deficiency
Biochimica et Biophysica Acta, 2011Co-Authors: Cristina Amat Di San Filippo, Nicola Longo, Orly ArdonAbstract:Abstract Primary Carnitine Deficiency is caused by impaired activity of the Na + -dependent OCTN2 Carnitine/organic cation transporter. Carnitine is essential for entry of long-chain fatty acids into mitochondria and its Deficiency impairs fatty acid oxidation. Most missense mutations identified in patients with primary Carnitine Deficiency affect putative transmembrane or intracellular domains of the transporter. Exceptions are the substitutions P46S and R83L located in an extracellular loop close to putative glycosylation sites (N57, N64, and N91) of OCTN2. P46S and R83L impaired glycosylation and maturation of OCTN2 transporters to the plasma membrane. We tested whether glycosylation was essential for the maturation of OCTN2 transporters to the plasma membrane. Substitution of each of the three asparagine (N) glycosylation sites with glutamine (Q) decreased Carnitine transport. Substitution of two sites at a time caused a further decline in Carnitine transport that was fully abolished when all three glycosylation sites were substituted by glutamine (N57Q/N64Q/N91Q). Kinetic analysis of Carnitine and sodium-stimulated Carnitine transport indicated that all substitutions decreased the Vmax for Carnitine transport, but N64Q/N91Q also significantly increased the Km toward Carnitine, indicating that these two substitutions affected regions of the transporter important for substrate recognition. Western blot analysis confirmed increased mobility of OCTN2 transporters with progressive substitutions of asparagines 57, 64 and/or 91 with glutamine. Confocal microscopy indicated that glutamine substitutions caused progressive retention of OCTN2 transporters in the cytoplasm, up to full retention (such as that observed with R83L) when all three glycosylation sites were substituted. Tunicamycin prevented OCTN2 glycosylation, but it did not impair maturation to the plasma membrane. These results indicate that OCTN2 is physiologically glycosylated and that the P46S and R83L substitutions impair this process. Glycosylation does not affect maturation of OCTN2 transporters to the plasma membrane, but the 3 asparagines that are normally glycosylated are located in a region important for substrate recognition and turnover rate.
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validation of dye binding high resolution thermal denaturation for the identification of mutations in the slc22a5 gene
Human Mutation, 2005Co-Authors: Steven F. Dobrowolski, Jason T. Mckinney, Cristina Amat Di San Filippo, K. G. Sim, Bridget Wilcken, Nicola LongoAbstract:Primary Carnitine Deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective Carnitine transport. This disease is caused by mutations in the OCTN2 Carnitine transporter encoded by the SLC22A5 gene. Here we validate dye-binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA binding dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the Carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary Carnitine Deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary Carnitine Deficiency and indicate dye-binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration. Hum Mutat 25:306–313, 2005. © 2005 Wiley-Liss, Inc.
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tyrosine residues affecting sodium stimulation of Carnitine transport in the octn2 Carnitine organic cation transporter
Journal of Biological Chemistry, 2004Co-Authors: Cristina Amat Di San Filippo, Nicola LongoAbstract:Abstract Primary Carnitine Deficiency is a disorder of fatty acid oxidation caused by mutations in the Na+-dependent Carnitine/organic cation transporter OCTN2. Studies with tyrosyl group-modifying reagents support the involvement of tyrosine residues in Na+ binding by sodium-coupled transporters. Here we report two new patients with Carnitine Deficiency caused by mutations affecting tyrosyl residues (Y447C and Y449D) close to a residue (Glu-452) previously shown to affect sodium stimulation of Carnitine transport. Kinetic analysis indicated that the Y449D substitution, when expressed in Chinese hamster ovary cells, increased the concentration of sodium required to half-maximally stimulate Carnitine transport from 14.8 ± 1.8 to 34.9 ± 5.8 mm (p < 0.05), whereas Y447C completely abolished Carnitine transport. Substitution of these tyrosine residues with phenylalanine restored normal Carnitine transport in Y449F but resulted in markedly impaired Carnitine transport by Y447F. This was associated with an increase in the concentration of sodium required to half-maximally stimulate Carnitine transport to 57.8 ± 7.4 mm (p < 0.01 versus normal OCTN2). The Y447F and Y449D mutant transporters retained their ability to transport the organic cation tetraethylammonium indicating that their effect on Carnitine transport was specific and likely associated with the impaired sodium stimulation of Carnitine transport. By contrast, the Y447C natural mutation abolished the transport of organic cations in addition to Carnitine. Confocal microscopy of OCTN2 transporters tagged with green fluorescent protein indicated that the Y447C mutant transporters failed to reach the plasma membrane, whereas Y447F, Y449D, and Y449F had normal membrane localization. These natural mutations identify tyrosine residues possibly involved in coupling the sodium electrochemical gradient to transmembrane solute transfer in the sodium-dependent co-transporter OCTN2.
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the octn2 Carnitine transporter and fatty acid oxidation
2003Co-Authors: Nicola Longo, Cristina Amat Di San Filippo, Marzia PasqualiAbstract:Carnitine (3-hydroxy -4-trimethylammonium butyrate) is a hydrophilic molecule that plays an essential role in the transfer of long-chain fatty acids into mitochondria for β-oxidation (Scaglia and Longo 1999). Carnitine also binds acyl residues and helps in their elimination. This decreases the number of acyl residues conjugated with Coenzyme A (CoA) and increases the ratio between free and acylated CoA (Bieber 1988). Less defined functions of Carnitine include the shuttling of fatty acids between different intracellular organelle s (peroxisomes, microsomes, and mitochondria) involved in fatty acid metabolism (Bieber 1988). Carnitine Deficiency has been known for several years in humans, but the difference between primary and secondary Carnitine Deficiency has only been fully defined in recent years. This chapter will review the structure and function of the OCTN2 Carnitine transporter defective in primary Carnitine Deficiency.
Vadivel Ganapathy - One of the best experts on this subject based on the ideXlab platform.
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na and cl coupled active transport of Carnitine by the amino acid transporter atb 0 from mouse colon expressed in hrpe cells and xenopus oocytes
The Journal of Physiology, 2001Co-Authors: Takeo Nakanishi, Frederick H Leibach, Wei Huang, Malliga E Ganapathy, Takahiro Hatanaka, Puttur D Prasad, Vadivel GanapathyAbstract:Carnitine (β-hydroxy-γ-trimethylaminobutyrate) is an obligate requirement for β-oxidation of long-chain fatty acids. It is synthesized endogenously in humans in the liver and kidney (Carter et al. 1995). It is also absorbed in the intestinal tract from dietary sources (Rebouche, 1992). The biological importance of this molecule is evident from the clinical consequences of Carnitine Deficiency encountered in a variety of genetic and acquired diseases (Kerner & Hoppel, 1998). The symptoms of Carnitine Deficiency include skeletal myopathy, cardiomyopathy, encephalopathy and failure to thrive (Treem et al. 1988; Kerner & Hoppel, 1998). Most tissues, including the cardiac and skeletal muscle, contain intracellular Carnitine levels severalfold higher than plasma levels due to the presence of a Na+-dependent high-affinity Carnitine transport system (Bremer, 1983). This transport system also exists in the brush border membrane of renal tubular epithelial cells where it plays a role in the reabsorption of Carnitine (Rebouche & Mack, 1984; Huang et al. 1999). A genetic defect in this transport system results in excessive urinary loss of Carnitine, causing systemic Carnitine Deficiency. Since the same transport system is also responsible for active accumulation of Carnitine in tissues such as the heart and skeletal muscle, the genetic defect is associated with drastically reduced intracellular levels of Carnitine in these tissues. The major clinical symptoms of this defect, known as primary Carnitine Deficiency, are skeletal and cardiac myopathy, resulting from impaired energy production from fatty acid oxidation as a consequence of reduced intracellular levels of Carnitine. Recently, this transporter has been cloned (Wu et al. 1998; Tamai et al. 1998). Interestingly, this transporter also transports several organic cations and β-lactam antibiotics (Wu et al. 1998, 1999; Ohashi et al. 1999; Ganapathy et al. 2000). Furthermore, it belongs to the organic cation transporter gene family on the basis of its primary structure (Wu et al. 1998). Therefore, the transporter is named OCTN2 (novel organic cation transporter 2). The present studies describe the identification of a second energy-coupled Carnitine transporter. This transporter, known as ATB0,+, is an amino acid transporter expressed in the intestine, lung and mammary gland. Functionally, ATB0,+ is a Na+- and Cl−-coupled transport system for neutral and cationic amino acids. It plays an important role in the absorption of amino acids in the intestinal tract (Ganapathy et al. 2001). The cloning of human ATB0,+ has been recently reported (Sloan & Mager, 1999). To date, the transport function of ATB0,+ has been studied only with amino acids as substrates. Its transport function is highly concentrative, energized by transmembrane gradients of Na+ and Cl− and membrane potential. ATB0,+ belongs to the gene family of Na+- and Cl−-coupled transporters for a variety of compounds such as amino acids (e.g. glycine and proline), neurotransmitters (e.g. monoamines and γ-aminobutyrate) and osmolytes (e.g. taurine and betaine). Structurally, ATB0,+ is very closely related to γ-aminobutyrate transporters and betaine transporter. Therefore, we tested whether ATB0,+ is able to recognize γ-aminobutyrate and other structurally related compounds as substrates. These studies have led to an interesting finding that ATB0,+ can transport Carnitine coupled to the transmembrane gradients of Na+ and Cl−.
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β lactam antibiotics as substrates for octn2 an organic cation Carnitine transporter
Journal of Biological Chemistry, 2000Co-Authors: Malliga E Ganapathy, Frederick H Leibach, Wei Huang, Prasanna D Rajan, Lee A Carter, Mitsuru Sugawara, Ken Iseki, Vadivel GanapathyAbstract:Therapeutic use of cephaloridine, a β-lactam antibiotic, in humans is associated with Carnitine Deficiency. A potential mechanism for the development of Carnitine Deficiency is competition between cephaloridine and Carnitine for the renal reabsorptive process. OCTN2 is an organic cation/Carnitine transporter that is responsible for Na+-coupled transport of Carnitine in the kidney and other tissues. We investigated the interaction of several β-lactam antibiotics with OCTN2 using human cell lines that express the transporter constitutively as well as using cloned human and rat OCTN2s expressed heterologously in human cell lines. The β-lactam antibiotics cephaloridine, cefoselis, cefepime, and cefluprenam were found to inhibit OCTN2-mediated Carnitine transport. These antibiotics possess a quaternary nitrogen as does Carnitine. Several other β-lactam antibiotics that do not possess this structural feature did not interact with OCTN2. The interaction of cephaloridine with OCTN2 is competitive with respect to Carnitine. Interestingly, many of the β-lactam antibiotics that were not recognized by OCTN2 were good substrates for the H+-coupled peptide transporters PEPT1 and PEPT2. In contrast, cephaloridine, cefoselis, cefepime, and cefluprenam, which were recognized by OCTN2, did not interact with PEPT1 and PEPT2. The interaction of cephaloridine with OCTN2 was Na+-dependent, whereas the interaction of cefoselis and cefepime with OCTN2 was largely Na+-independent. Furthermore, the Na+-dependent, OCTN2-mediated cellular uptake of cephaloridine could be demonstrated by direct uptake measurements. These studies show that OCTN2 plays a crucial role in the pharmacokinetics and therapeutic efficacy of certain β-lactam antibiotics such as cephaloridine and that cephaloridine-induced Carnitine Deficiency is likely to be due to inhibition of Carnitine reabsorption in the kidney.
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mutations in novel organic cation transporter octn2 an organic cation Carnitine transporter with differential effects on the organic cation transport function and the Carnitine transport function
Journal of Biological Chemistry, 1999Co-Authors: Pankaj Seth, Frederick H Leibach, Wei Huang, Vadivel GanapathyAbstract:Abstract Novel organic cation transporter (OCTN2) is an organic cation/Carnitine transporter, and two missense mutations, L352R and P478L, in OCTN2 have been identified as the cause for primary Carnitine Deficiency. In the present study, we assessed the influence of these two mutations on the Carnitine transport function and the organic cation transport function of OCTN2. The L352R mutation resulted in a complete loss of both transport functions. In contrast, the P478L mutation resulted in a complete loss of only the Carnitine transport function but significantly stimulated the organic cation transport function. Studies with human OCTN2/rat OCTN2 chimeric transporters indicated that the Carnitine transport site and the organic cation transport site were not identical. Because Carnitine transport is Na+-dependent whereas organic cation transport is Na+-independent, we investigated the possibility that the P478L mutation affected Na+ binding. The Na+ activation kinetics were found to be similar for the P478L mutant and wild type OCTN2. We then mutated nine different tyrosine residues located in or near transmembrane domains and assessed the transport function of these mutants. One of these mutations, Y211F, was found to have differential influence on the two transport activities of OCTN2 as did the P478L mutation. However, the Na+ activation kinetics were not affected. These findings are of clinical relevance to patients with primary Carnitine Deficiency because whereas each and every mutation in these patients is expected to result in the loss of the Carnitine transport function, all of these mutations may not interfere with the organic cation transport function.
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mutations of octn2 an organic cation Carnitine transporter lead to deficient cellular Carnitine uptake in primary Carnitine Deficiency
Human Molecular Genetics, 1999Co-Authors: Nelson L S Tang, Pankaj Seth, Vadivel Ganapathy, Joannie Hui, Patrick Man Pan Yuen, Tai Fai Fok, N M HjelmAbstract:Systemic primary Carnitine Deficiency (CDSP, OMIM 212140) is an autosomal recessive disease characterized by low serum and intracellular concentrations of Carnitine. CDSP may present with acute metabolic derangement simulating Reye's syndrome within the first 2 years of life. After 3 years of age, patients with CDSP may present with cardiomyopathy and muscle weakness. A linkage with D5S436 in 5q was reported in a family. A recently cloned homologue of the organic cation transporter, OCTN2, which has sodium-dependent Carnitine uptake properties, was also mapped to the same locus. We screened for mutation in OCTN2 in a confirmed CDSP family. One truncating mutation (Trp132Stop) and one missense mutation (Pro478Leu) of OCTN2 were identified together with two silent polymorphisms. Expression of the mutant cDNAs revealed virtually no uptake activity for both mutations. Our data indicate that mutations in OCTN2 are responsible for CDSP. Identification of the underlying gene in this disease will allow rapid detection of carriers and postnatal diagnosis of affected patients.
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mutations in the organic cation Carnitine transporter octn2 in primary Carnitine Deficiency
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Yuhuan Wang, Vadivel Ganapathy, Jing Ye, Nicola LongoAbstract:Primary Carnitine Deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective Carnitine transport. This disease presents early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. The gene for this condition maps to 5q31.2–32 and OCTN2, an organic cation/Carnitine transporter, also maps to the same chromosomal region. Here we test the causative role of OCTN2 in primary Carnitine Deficiency by searching for mutations in this gene in affected patients. Fibroblasts from patients with primary Carnitine Deficiency lacked mediated Carnitine transport. Transfection of patient’s fibroblasts with the OCTN2 cDNA partially restored Carnitine transport. Sequencing of the OCTN2 gene revealed different mutations in two unrelated patients. The first patient was homozygous (and both parents heterozygous) for a single base pair substitution converting the codon for Arg-282 to a STOP codon (R282X). The second patient was a compound heterozygote for a paternal 1-bp insertion producing a STOP codon (Y401X) and a maternal 1-bp deletion that produced a frameshift creating a subsequent STOP codon (458X). These mutations decreased the levels of mature OCTN2 mRNA and resulted in nonfunctional transporters, confirming that defects in the organic cation/Carnitine transporter OCTN2 are responsible for primary Carnitine Deficiency.
Akira Tsuji - One of the best experts on this subject based on the ideXlab platform.
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molecular and functional characterization of organic cation Carnitine transporter family in mice
Journal of Biological Chemistry, 2000Co-Authors: Rikiya Ohashi, Junichi Nezu, Miyuki Shimane, Daisuke Kobayashi, Akira TsujiAbstract:Abstract Carnitine is essential for β-oxidation of fatty acids, and a defect of cell membrane transport of Carnitine leads to fatal systemic Carnitine Deficiency. We have already shown that a defect of the organic cation/Carnitine transporter OCTN2 is a primary cause of systemic Carnitine Deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for Carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na+-dependent, whereas that by OCTN3 was Na+-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of Carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for Carnitine and significantly lower Carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na+-independent Carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of Carnitine transport activity and may have a different role from other members of the OCTN family.
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molecular and functional characterization of organic cation Carnitine transporter family in mice
Journal of Biological Chemistry, 2000Co-Authors: Ikumi Tamai, Rikiya Ohashi, Junichi Nezu, Miyuki Shimane, Daisuke Kobayashi, Yoshimichi Sai, Asuka Oku, Akira TsujiAbstract:Carnitine is essential for beta-oxidation of fatty acids, and a defect of cell membrane transport of Carnitine leads to fatal systemic Carnitine Deficiency. We have already shown that a defect of the organic cation/Carnitine transporter OCTN2 is a primary cause of systemic Carnitine Deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for Carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na(+)-dependent, whereas that by OCTN3 was Na(+)-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of Carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for Carnitine and significantly lower Carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na(+)-independent Carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of Carnitine transport activity and may have a different role from other members of the OCTN family.
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na dependent Carnitine transport by organic cation transporter octn2 its pharmacological and toxicological relevance
Journal of Pharmacology and Experimental Therapeutics, 1999Co-Authors: Rikiya Ohashi, Junichi Nezu, Miyuki Shimane, Hikaru Yabuuchi, Yoshimichi Sai, Asuka Oku, Akira TsujiAbstract:Carnitine Deficiency, either primary or drug-induced, causes critical symptoms and is thought to involve alteration of active transport of Carnitine across the plasma membrane of tissues as the underlying mechanism. Recently, we showed that human organic cation transporter, hOCTN2, cloned as a member of the organic cation transporter family, is a physiologically important Na+-dependent high-affinity Carnitine transporter in humans. In this study, we further characterized the functional properties of hOCTN2 and examined the interaction between hOCTN2-mediated Carnitine transport and clinically used drugs to assess possible toxicological effects. When expressed in human embryonic kidney (HEK)293 cells, hOCTN2 showed low but significant stereospecific transport activity:d-Carnitine was transported with lower affinity ( Km = 10.9 μM) than thel-isomer ( Km = 4.3 μM). One Na+ appeared to be associated with the transport of one Carnitine molecule. hOCTN2-mediated transport of acetyl-l-Carnitine was also Na+-dependent and of high affinity, with a Km value of 8.5 μM. To examine the transport activity for organic cations other than Carnitine and the possible relationship of drug-induced Carnitine Deficiency with hOCTN2, the inhibitory effect of several drugs on hOCTN2-mediated l-Carnitine transport was examined. Many zwitterionic drugs, such as cephaloridine, and many cationic drugs, such as quinidine and verapamil, exhibited significant inhibitory effects. Among these inhibitors, tetraethylammonium, pyrilamine, quinidine, verapamil, and valproate were found to be transported by hOCTN2. The results suggest that the Carnitine Deficiency-related toxicological effects by long-term treatment with such drugs might be ascribed to a functional alteration of hOCTN2-mediated Carnitine transport.
Rikiya Ohashi - One of the best experts on this subject based on the ideXlab platform.
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molecular and functional characterization of organic cation Carnitine transporter family in mice
Journal of Biological Chemistry, 2000Co-Authors: Rikiya Ohashi, Junichi Nezu, Miyuki Shimane, Daisuke Kobayashi, Akira TsujiAbstract:Abstract Carnitine is essential for β-oxidation of fatty acids, and a defect of cell membrane transport of Carnitine leads to fatal systemic Carnitine Deficiency. We have already shown that a defect of the organic cation/Carnitine transporter OCTN2 is a primary cause of systemic Carnitine Deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for Carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na+-dependent, whereas that by OCTN3 was Na+-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of Carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for Carnitine and significantly lower Carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na+-independent Carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of Carnitine transport activity and may have a different role from other members of the OCTN family.
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molecular and functional characterization of organic cation Carnitine transporter family in mice
Journal of Biological Chemistry, 2000Co-Authors: Ikumi Tamai, Rikiya Ohashi, Junichi Nezu, Miyuki Shimane, Daisuke Kobayashi, Yoshimichi Sai, Asuka Oku, Akira TsujiAbstract:Carnitine is essential for beta-oxidation of fatty acids, and a defect of cell membrane transport of Carnitine leads to fatal systemic Carnitine Deficiency. We have already shown that a defect of the organic cation/Carnitine transporter OCTN2 is a primary cause of systemic Carnitine Deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for Carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na(+)-dependent, whereas that by OCTN3 was Na(+)-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of Carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for Carnitine and significantly lower Carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na(+)-independent Carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of Carnitine transport activity and may have a different role from other members of the OCTN family.
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na dependent Carnitine transport by organic cation transporter octn2 its pharmacological and toxicological relevance
Journal of Pharmacology and Experimental Therapeutics, 1999Co-Authors: Rikiya Ohashi, Junichi Nezu, Miyuki Shimane, Hikaru Yabuuchi, Yoshimichi Sai, Asuka Oku, Akira TsujiAbstract:Carnitine Deficiency, either primary or drug-induced, causes critical symptoms and is thought to involve alteration of active transport of Carnitine across the plasma membrane of tissues as the underlying mechanism. Recently, we showed that human organic cation transporter, hOCTN2, cloned as a member of the organic cation transporter family, is a physiologically important Na+-dependent high-affinity Carnitine transporter in humans. In this study, we further characterized the functional properties of hOCTN2 and examined the interaction between hOCTN2-mediated Carnitine transport and clinically used drugs to assess possible toxicological effects. When expressed in human embryonic kidney (HEK)293 cells, hOCTN2 showed low but significant stereospecific transport activity:d-Carnitine was transported with lower affinity ( Km = 10.9 μM) than thel-isomer ( Km = 4.3 μM). One Na+ appeared to be associated with the transport of one Carnitine molecule. hOCTN2-mediated transport of acetyl-l-Carnitine was also Na+-dependent and of high affinity, with a Km value of 8.5 μM. To examine the transport activity for organic cations other than Carnitine and the possible relationship of drug-induced Carnitine Deficiency with hOCTN2, the inhibitory effect of several drugs on hOCTN2-mediated l-Carnitine transport was examined. Many zwitterionic drugs, such as cephaloridine, and many cationic drugs, such as quinidine and verapamil, exhibited significant inhibitory effects. Among these inhibitors, tetraethylammonium, pyrilamine, quinidine, verapamil, and valproate were found to be transported by hOCTN2. The results suggest that the Carnitine Deficiency-related toxicological effects by long-term treatment with such drugs might be ascribed to a functional alteration of hOCTN2-mediated Carnitine transport.
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Primary systemic Carnitine Deficiency is caused by mutations in a gene encoding sodium ion-dependent Carnitine transporter
Nature genetics, 1999Co-Authors: Junichi Nezu, Rikiya Ohashi, Hiroko Nikaido, Hikaru Yabuuchi, Ikumi Tamai, Yoshimichi Sai, Asuka Oku, Akio Koizumi, Noriyoshi Hashimoto, Yutaka ShojiAbstract:Primary systemic Carnitine Deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia1,2,3. SCD has also been linked to sudden infant death syndrome4. Membrane-physiological studies have suggested a defect of the Carnitine transport system in the plasma membrane in SCD patients5 and in the mouse model, juvenile visceral steatosis ( jvs; ref. 6 ). Although the responsible loci have been mapped in both human7 and mouse8, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2 ( ref. 9 ). Our observation that OCTN2 has the ability to transport Carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5 . Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates Carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.
Ikumi Tamai - One of the best experts on this subject based on the ideXlab platform.
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pharmacological and pathophysiological roles of Carnitine organic cation transporters octns slc22a4 slc22a5 and slc22a21
Biopharmaceutics & Drug Disposition, 2013Co-Authors: Ikumi TamaiAbstract:The Carnitine/organic cation transporter (OCTN) family consists of three transporter isoforms, i.e. OCTN1 (SLC22A4) and OCTN2 (SLC22A5) in humans and animals and Octn3 (Slc22a21) in mice. These transporters are physiologically essential to maintain appropriate systemic and tissue concentrations of Carnitine by regulating its membrane transport during intestinal absorption, tissue distribution and renal reabsorption. Among them, OCTN2 is a sodium-dependent, high-affinity transporter of Carnitine, and a functional defect of OCTN2 due to genetic mutation causes primary systemic Carnitine Deficiency (SCD). Since Carnitine is essential for beta-oxidation of long-chain fatty acids to produce ATP, OCTN2 gene mutation causes a range of symptoms, including cardiomyopathy, skeletal muscle weakness, fatty liver and male infertility. These functional consequences of Octn2 gene mutation can be seen clearly in an animal model, jvs mouse, which exhibits the SCD phenotype. In addition, although the mechanism is not clear, single nucleotide polymorphisms of OCTN1 and OCTN2 genes are associated with increased incidences of rheumatoid arthritis, Crohn's disease and asthma. OCTN1 and OCTN2 accept cationic drugs as substrates and contribute to intestinal and pulmonary absorption, tissue distribution (including to tumour cells), and renal excretion of these drugs. Modulation of the transport activity of OCTN2 by externally administered drugs may cause drug-induced secondary Carnitine Deficiency. Rodent Octn3 transports Carnitine specifically, particularly in male reproductive tissues. Thus, the OCTNs are physiologically, pathologically and pharmacologically important. Detailed characterization of these transporters will greatly improve our understanding of the pathology associated with common diseases caused by functional Deficiency of OCTNs.
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molecular and functional characterization of organic cation Carnitine transporter family in mice
Journal of Biological Chemistry, 2000Co-Authors: Ikumi Tamai, Rikiya Ohashi, Junichi Nezu, Miyuki Shimane, Daisuke Kobayashi, Yoshimichi Sai, Asuka Oku, Akira TsujiAbstract:Carnitine is essential for beta-oxidation of fatty acids, and a defect of cell membrane transport of Carnitine leads to fatal systemic Carnitine Deficiency. We have already shown that a defect of the organic cation/Carnitine transporter OCTN2 is a primary cause of systemic Carnitine Deficiency. In the present study, we further isolated and characterized new members of the OCTN family, OCTN1 and -3, in mice. All three members were expressed commonly in kidney, and OCTN1 and -2 were also expressed in various tissues, whereas OCTN3 was characterized by predominant expression in testis. When their cDNAs were transfected into HEK293 cells, the cells exhibited transport activity for Carnitine and/or the organic cation tetraethylammonium (TEA). Carnitine transport by OCTN1 and OCTN2 was Na(+)-dependent, whereas that by OCTN3 was Na(+)-independent. TEA was transported by OCTN1 and OCTN2 but not by OCTN3. The relative uptake activity ratios of Carnitine to TEA were 1.78, 11.3, and 746 for OCTN1, -2, and -3, respectively, suggesting high specificity of OCTN3 for Carnitine and significantly lower Carnitine transport activity of OCTN1. Thus, OCTN3 is unique in its limited tissue distribution and Na(+)-independent Carnitine transport, whereas OCTN1 efficiently transported TEA with minimal expression of Carnitine transport activity and may have a different role from other members of the OCTN family.
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Primary systemic Carnitine Deficiency is caused by mutations in a gene encoding sodium ion-dependent Carnitine transporter
Nature genetics, 1999Co-Authors: Junichi Nezu, Rikiya Ohashi, Hiroko Nikaido, Hikaru Yabuuchi, Ikumi Tamai, Yoshimichi Sai, Asuka Oku, Akio Koizumi, Noriyoshi Hashimoto, Yutaka ShojiAbstract:Primary systemic Carnitine Deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia1,2,3. SCD has also been linked to sudden infant death syndrome4. Membrane-physiological studies have suggested a defect of the Carnitine transport system in the plasma membrane in SCD patients5 and in the mouse model, juvenile visceral steatosis ( jvs; ref. 6 ). Although the responsible loci have been mapped in both human7 and mouse8, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2 ( ref. 9 ). Our observation that OCTN2 has the ability to transport Carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5 . Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates Carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.