Primary carnitine deficiency, because of a defect of the tissue plasma membrane carnitine transporters, causes critical symptoms. However, the transporter has not been molecularly identified. In this study, we screened a human kidney cDNA library and assembled a cDNA-encoding OCTN2 as a homologue of the organic cation transporter OCTN1, and then we examined the function of OCTN2 as a carnitine transporter. OCTN2-cDNA encodes a polypeptide of 557 amino acids with 75.8% similarity to OCTN1. Northern blot analysis showed that OCTN2 is strongly expressed in kidney, skeletal muscle, heart, and placenta in adult humans. When OCTN2 was expressed in HEK293 cells, uptake of L-[ 3 H]carnitine was strongly enhanced in a sodium-dependent manner with K m value of 4.34 M, whereas typical substrates for previously known organic cation transporters, tetraethylammonium and guanidine, were not good substitutes. OCTN2-mediated L-[ 3 H]carnitine transport was inhibited by the D-isomer, acetyl-D,Lcarnitine, and ␥-butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline, ␤-hydroxybutyric acid, ␥-aminobutyric acid, lysine, and taurine were not inhibitory. Because the observed tissue distribution of OCTN2 is consistent with the reported distribution of carnitine transport activity and the functional characteristics of OCTN2 coincide with those reported for plasma membrane carnitine transport, we conclude that OCTN2 is a physiologically important, high affinity sodium-carnitine cotransporter in humans.

Carnitine is essential for ␤-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.

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis. Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2. 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.

OCTN2 is an Na(+)-dependent transporter for carnitine, which is essential for fatty acid metabolism, and its functional defect leads to fatal systemic carnitine deficiency (SCD). It also transports the organic cation tetraethylammonium (TEA) in an Na(+)-independent manner. Here, we studied the multifunctionality of OCTN2, by examining the transport characteristics in cells transfected with mouse OCTN2 and in juvenile visceral steatosis (jvs) mice that exhibit a SCD phenotype owing to mutation of the OCTN2 gene. The physiological significance of OCTN2 as an organic cation transporter was confirmed by using jvs mice. The embryonic fibroblasts from jvs mice exhibited significantly decreased transport of [(14)C]TEA. Pharmacokinetic analysis of [(14)C]TEA disposition demonstrated that jvs mice showed decreased tissue distribution and renal secretory clearance. In transport experiments using OCTN2-expressing cells, TEA and carnitine showed mutual trans-stimulation effects in their transport, implying a carnitine/TEA exchange mechanism. In addition, Na(+) affected the affinity of carnitine for OCTN2, whereas Na(+) is unlikely to be involved in TEA transport. This is the first molecular and physiological demonstration of the operation of an organic cation transporter in renal apical membrane. The results are consistent with the physiological coupling of carnitine reabsorption with the secretion of organic cations.

Aggregation of the 42-residue amyloid beta-protein (Abeta42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Despite numerous structural studies on Abeta aggregates, the relationship between tertiary structure and toxicity remains unclear. Our proline scanning and solid-state NMR studies suggested that aggregates both of wild-type Abeta42 and of E22K-Abeta42 (one of the mutants related to cerebral amyloid angiopathy) contain two conformers: a major one with a turn at positions 25 and 26, and a minor one with a turn at positions 22 and 23. To identify the toxic conformer, the derivative Abeta42-lactam(22K-23E), in which the side chains at positions 22 and 23 were covalently linked, was synthesized as a minor conformer surrogate, along with Abeta42-lactam(25K-26E) as a major conformer surrogate. The Abeta42-lactam(22K-23E) showed stronger aggregation, neurotoxicity, radical generation, and oligomerization than wild-type Abeta42, whereas in Abeta42-lactam(25K-26E) were weak. The transition from the physiological conformation with a turn at positions 25 and 26 to the toxic conformation with a turn at positions 22 and 23 might be a key event in the pathogenesis of AD.

Serum free-carnitine levels were determined in 973 unrelated white collar workers in Akita, Japan. Fourteen of these participants consistently had serum free-carnitine levels below the fifth percentile (28 microM for females and 38 microM for males). The OCTN2 (organic cation transporter) gene was sequenced for these 14 subjects, for 22 subjects whose carnitine levels were below the fifth percentile in the first screening but were normal in the second measurement and in 69 individuals with normal carnitine levels for two separate measurements. Polymorphic sequences defined three major haplotypes with equal frequency. Mutations were identified in nine subjects with low carnitine levels: Trp132X (three individuals), Ser467Cys (four), Trp283Cys (one) and Met179Leu (one). In vitro expression studies in HEK cells indicated that Ser467Cys and Trp283Cys, but not Met179Leu, significantly reduced L-carnitine uptake relative to the normal control. Trp132X and Ser467Cys were associated with specific haplotypes, suggesting a founder effect. A conservative estimate of the overall prevalence of heterozygotes was 1.01% in the Akita prefecture, Japan, giving an estimated incidence of primary systemic carnitine deficiency (MIM 212140) as 1 in 40 000 births. An echocardiographic study of the families of patients with primary carnitine deficiency revealed that the heterozygotes for OCTN2 mutations were predisposed to late onset benign cardiac hypertrophy (odds ratio 15.1, 95% CI 1.39-164) compared with the wild-types. Sequencing of DNA isolated from three deceased siblings (1.5-8 years) in two families retrospectively confirmed that all three deceased subjects were homozygous for the OCTN2 mutations.

Predicting the fraction unbound in plasma provides a good understanding of the pharmacokinetic properties of a drug to assist candidate selection in the early stages of drug discovery. It is also an effective tool to mitigate the risk of late-stage attrition and to optimize further screening. In this study, we built in silico prediction models of fraction unbound in human plasma with freely available software, aiming specifically to improve the accuracy in the low value ranges. We employed several machine learning techniques and built prediction models trained on the largest ever data set of 2738 experimental values. The classification model showed a high true positive rate of 0.826 for the low fraction unbound class on the test set. The strongly biased distribution of the fraction unbound in plasma was mitigated by a logarithmic transformation in the regression model, leading to improved accuracy at lower values. Overall, our models showed better performance than those of previously published methods, including commercial software. Our prediction tool can be used on its own or integrated into other pharmacokinetic modeling systems.

ABSTRACT:In the present study, we examined in vitro biliary clearance of several compounds in sandwich-cultured rat hepatocytes (SCRH) and compared it with that observed in vivo in rats; the effect of plasma protein binding on in vitro-in vivo correlation of biliary excretion was also assessed. The in vitro biliary excretion was determined by differential cumulative uptake of compounds in SCRH preincubated in the presence and absence of Ca 2؉ /Mg 2؉ .The cumulative uptake study of radiolabeled substrates revealed that the function of canalicular efflux transporters such as bile salt export pump, multidrug resistance-associated protein 2, breast cancer resistance protein, and multidrug resistance 1 was adequately maintained in SCRH. Unlabeled test compounds, pravastatin, rosuvastatin, valsartan, cefmetazole, and cefoperazone exhibited varying degrees of in vitro biliary excretion in the cumulative uptake study using SCRH. In vivo biliary excretions of these compounds were measured in common bile duct-cannulated rats. Whereas their biliary excretion ratios were all more than 60% of the dose, the in vivo intrinsic biliary clearances varied from 10.5 to 1787.2 ml/min/kg. The in vitro intrinsic biliary clearances of test compounds were well correlated with their corresponding in vivo intrinsic clearances calculated on the basis of the plasma unbound concentration (r 2 ‫؍‬ 0.984), whereas less correlation was observed when they were calculated on the basis of plasma total concentration (r 2 ‫؍‬ 0.217). These results indicate that SCRH is a useful in vitro model for predicting in vivo intrinsic biliary clearance in rats. In addition, for an accurate prediction, it is necessary to evaluate the in vivo intrinsic biliary clearance based on plasma unbound concentration but not total concentration.