The role of N-glycosylation in transport to the plasma membrane and sorting of the neuronal glycine transporter GLYT2

Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35-40% after enzymatic deglycosylation of the transporter reconstituted into liposomes. Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellular loop of GLYT2, produced an inactive protein that was retained in intracellular compartments when transiently transfected in COS cells or in nonpolarized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 localizes in the apical surface as assessed by transport and biotinylation assays. However, a partially unglycosylated mutant (triple mutant) was distributed in a nonpolarized manner in MDCK cells. The apical localization of GLYT2 occurred by a glycolipid rafts independent pathway.     

Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2

The neurotransmitter glycine is removed from the synaptic cleft by two Na(+)-and Cl(-)-dependent transporters, the glial (GLYT1) and neuronal (GLYT2) glycine transporters. GLYT2 lacks a conserved cysteine in the first hydrophilic loop (EL1) that is reactive to [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) in related transporters. A chimeric GLYT2 (GLYT2a-EL1) that contains GLYT1 sequences in this region, including the relevant cysteine, was sensitive to the reagent, and its sensitivity was decreased by co-substrates. We combined cysteine-specific biotinylation to detect transporter-reagent interactions with MTSET inactivation assays and temperature dependence analysis to study the mechanism by which Cl(-), Na(+), and glycine reduce methanethiosulfonate reagent inhibition. We demonstrate a Na(+) protective effect rather than an increased susceptibility to the reagent exerted by Li(+), as reported for the serotonin transporter. The different inhibition, protection, and reactivation properties between GLYT2a-EL1 and serotonin transporter suggest that EL1 is a source of structural heterogeneity involved in the specific effect of lithium on serotonin transport. The protection by Na(+) or Cl(-) on GLYT2a-EL1 was clearly dependent on temperature, suggesting that EL1 is not involved in ion binding but is subjected to ion-induced conformational changes. Na(+) and Cl(-) were required for glycine protection, indicating the necessity of prior ion interaction with the transporter for the binding of glycine. We conclude that EL1 acts as a fluctuating hinge undergoing sequential conformational changes during the transport cycle.     

Calcium- and syntaxin 1-mediated trafficking of the neuronal glycine transporter GLYT2

Previously we demonstrated the existence of a physical and functional interaction between the glycine transporters and the SNARE protein syntaxin 1. In the present report the physiological role of the syntaxin 1-glycine transporter 2 (GLYT2) interaction has been investigated by using a brain-derived preparation. Previous studies, focused on syntaxin 1-transporter interactions using overexpression systems, led to the postulation that syntaxin is somehow implicated in protein trafficking. Since syntaxin 1 is involved in exocytosis of neurotransmitter and also interacts with GLYT2, we stimulated exocytosis in synaptosomes and examined its effect on surface-expression and transport activity of GLYT2. We found that, under conditions that stimulate vesicular glycine release, GLYT2 is rapidly trafficked first toward the plasma membrane and then internalized. When the same experiments were performed with synaptosomes inactivated for syntaxin 1 by a pretreatment with the neurotoxin Bont/C, GLYT2 was unable to reach the plasma membrane but still was able to leave it. These results indicate the existence of a SNARE-mediated regulatory mechanism that controls the surface-expression of GLYT2. Syntaxin 1 is involved in the arrival to the plasma membrane but not in the retrieval. Furthermore, by using immunogold labeling on purified preparations from synaptosomes, we demonstrate that GLYT2 is present in small synaptic-like vesicles. GLYT2-containing vesicles may represent neurotransmitter transporter that is being trafficked. The results of our work suggest a close correlation between exocytosis of neurotransmitter and its reuptake by transporters.     

Localization of the glycine transporter GLYT1 in glutamatergic synaptic vesicles

We have previously shown the presence of the glycine transporter GLYT1 in glutamatergic terminals of the rat brain. In this study we present immunohistochemical and biochemical evidence indicating that GLYT1 is expressed not only at the plasma membrane of glutamatergic neurons, but also at synaptic vesicles. Confocal microscopy, immunoblots analysis of a highly purified synaptic vesicle fraction and immunoisolation of synaptic vesicles with anti-synaptophysin antibodies strongly suggested the presence of GLYT1 in synaptic vesicles. Moreover, direct observation with the electron microscope of purified vesicles immunoreacted with anti-GLYT1 and colloidal gold demonstrated that about 40% of the small vesicles of the purified vesicle fraction contained GLYT1. Double labeling for GLYT1 and synaptophysin of this vesicular fraction revealed that more of ninety percent of them were synaptic vesicles. Moreover, a significant part of the GLYT1 containing vesicles (86%) also contained the vesicular glutamate transporter vGLUT1, suggesting a functional role of GLYT1 in a subpopulation of glutamatergic vesicles.     

Transmembrane domain III plays an important role in ion binding and permeation in the glycine transporter GLYT2

The neuronal glycine transporter GLYT2 takes up glycine from the extracellular space by an electrogenic process where this neurotransmitter is co-transported with sodium and chloride ions. We report in this paper that tyrosine at position 289 of GLYT2a is crucial for ion coupling, glycine affinity and sodium selectivity, stressing the essential role played by this residue of transmembrane domain III in the mechanism of transport. Substitution to tryptophan (Y289W), phenylalanine (Y289F), or serine (Y289S), renders transporters unable to catalyze glycine uptake. Measurements of glycine evoked steady-state currents in transfected HEK-293 cells reveal EC(50) values for glycine 17-fold (Y289F) and 45-fold (Y289S) higher than that of the wild type transporter. Sodium dependence is severely altered in tyrosine 289 mutants, both at the level of apparent affinity and cooperativity, with the more dramatic change corresponding to the less conservative substitution (Y289S). Accordingly, sodium selectivity is gradually lost in Y289F and Y289S mutants, and chloride dependence of glycine evoked currents is markedly decreased in Y289F and Y289S mutants. In the absence of three-dimensional information from these transporters, these results provide experimental evidence supporting the hypothesis of transmembrane domain III being part of a common permeation pathway for substrate and co-transported ions.     

Molecular basis of the differential interaction with lithium of glycine transporters GLYT1 and GLYT2

Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na(+)/Cl(-)/glycine cotransport. GLYT1 displays a 2:1 :1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3:1 :1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na(+)-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2.     

Mutation of amino acid 475 of rat organic cation transporter 2 (rOCT2) impairs organic cation transport

Protein sequence alignments revealed one amino acid position, where organic cation transporters (OCTs, aspartate (D) at position 475 of rOCT2) and organic anion transporters (OATs, arginine (R) at position 466 of rOAT1) are charged oppositely. To address the impact of this amino acid for protein function we cloned rat organic cation transporter 2 (rOCT2), the renal electrogenic cation transporter of the basolateral side of proximal tubule cells. Site-directed mutagenesis was used to generate rOCT2-D475R (rOCT2-mut). Heterologous expression of rOCT2 wild-type (rOCT2-wt) in A6 cells resulted in a significant uptake of the fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP(+)). Accordingly, rOCT2-wt-transfected COS 7 cells showed an almost fourfold uptake of 25 microM [(14)C]-TEA, whereas rOCT2-mut did not exhibit any uptake of [(14)C]-TEA. These data indicate that rOCT2 transports both ASP(+) and TEA and that aspartate at position 475 of rOCT2 plays a critical role in transport function.     

Endocytosis of the neuronal glycine transporter GLYT2: role of membrane rafts and protein kinase C-dependent ubiquitination

Glycinergic neurotransmission is terminated by sodium- and chloride-dependent plasma membrane transporters. The neuronal glycine transporter 2 (GLYT2) supplies the terminal with substrate to refill synaptic vesicles containing glycine. This crucial process is defective in human hyperekplexia, a condition that can be caused by mutations in GLYT2. Inhibitory glycinergic neurotransmission is modulated by the GLYT2 exocytosis/endocytosis equilibrium, although the mechanisms underlying the turnover of this transporter remain elusive. We studied GLYT2 internalization pathways and the role of ubiquitination and membrane raft association of the transporter in its endocytosis. Using pharmacological tools, dominant-negative mutants and small-interfering RNAs, we show that the clathrin-mediated pathway is the primary mechanism for constitutive and regulated GLYT2 endocytosis in heterologous cells and neurons. We show that GLYT2 is constitutively internalized from cell surface lipid rafts, remaining associated with rafts in subcellular recycling structures. Protein kinase C (PKC) negatively modulates GLYT2 via rapid and dynamic redistribution of GLYT2 from raft to non-raft membrane subdomains and increasing ubiquitinated GLYT2 endocytosis. This biphasic mechanism is a versatile means to modulate GLYT2 behavior and hence, inhibitory glycinergic neurotransmission. These findings may reveal new therapeutic targets to address glycinergic pathologies associated with alterations in GLYT2 trafficking.     

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

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.     

Conserved asparagine residue located in binding pocket controls cation selectivity and substrate interactions in neuronal glutamate transporter

Transporters of the major excitatory neurotransmitter glutamate play a crucial role in glutamatergic neurotransmission by removing their substrate from the synaptic cleft. The transport mechanism involves co-transport of glutamic acid with three Na(+) ions followed by countertransport of one K(+) ion. Structural work on the archeal homologue Glt(Ph) indicates a role of a conserved asparagine in substrate binding. According to a recent proposal, this residue may also participate in a novel Na(+) binding site. In this study, we characterize mutants of this residue from the neuronal transporter EAAC1, Asn-451. None of the mutants, except for N451S, were able to exhibit transport. However, the K(m) of this mutant for l-aspartate was increased ～30-fold. Remarkably, the increase for d-aspartate and l-glutamate was 250- and 400-fold, respectively. Moreover, the cation specificity of N451S was altered because sodium but not lithium could support transport. A similar change in cation specificity was observed with a mutant of a conserved threonine residue, T370S, also implicated to participate in the novel Na(+) site together with the bound substrate. In further contrast to the wild type transporter, only l-aspartate was able to activate the uncoupled anion conductance by N451S, but with an almost 1000-fold reduction in apparent affinity. Our results not only provide experimental support for the Na(+) site but also suggest a distinct orientation of the substrate in the binding pocket during the activation of the anion conductance.     

A conserved aspartate residue located at the extracellular end of the binding pocket controls cation interactions in brain glutamate transporters

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.     

Mechanism of electrogenic cation transport by the cloned organic cation transporter 2 from rat

The organic cation transporter 2 (OCT2) is expressed in plasma membranes of kidney and brain. Its transport mechanism and substrates are debated. We studied substrate-induced changes of electrical current with the patch clamp technique after expression of rat OCT2 in oocytes. Activation of current, corresponding to efflux, was observed for small organic cations, e.g. choline. In contrast, the bigger cations quinine and tetrabutylammonium elicited no change in current. However, transport of choline could be inhibited by applying quinine or tetrabutylammonium to the cytoplasmic side. Inhibition of organic cation efflux by quinine was competitive with substrates. Quinine at the inside also inhibited substrate influx from the outside. Current-voltage analysis showed that both maximal turnover and apparent affinity to substrates are voltage-dependent. Substrate-induced currents with organic cations on both membrane sides reversed as predicted from the Nernst potential. Our results clearly identify the electrochemical potential as driving force for transport at neutral pH and exclude an electroneutral H(+)/organic cation(+) exchange. We suggest the existence of an electroneutral organic cation(+) exchange and propose a model for a carrier-type transport mechanism.     

High affinity transport of taurine by the Drosophila aspartate transporter dEAAT2

Excitatory amino acid transporters (EAATs) are structurally related plasma membrane proteins known to mediate the Na(+)/K(+)-dependent uptake of the amino acids l-glutamate and dl-aspartate. In the nervous system, these proteins contribute to the clearance of glutamate from the synaptic cleft and maintain excitatory amino acid concentrations below excitotoxic levels. Two homologues exist in Drosophila melanogaster, dEAAT1 and dEAAT2, which are specifically expressed in the nervous tissue. We previously reported that dEAAT2 shows unique substrate discrimination as it mediates high affinity transport of aspartate but not glutamate. We now show that dEAAT2 can also transport the amino acid taurine with high affinity, a property that is not shared by two other transporters of the same family, Drosophila dEAAT1 and human hEAAT2. Taurine transport by dEAAT2 was efficiently blocked by an EAAT antagonist but not by inhibitors of the structurally unrelated mammalian taurine transporters. Taurine and aspartate are transported with similar K(m) and relative efficacy and behave as mutually competitive inhibitors. dEAAT2 can mediate either net uptake or the heteroexchange of its two substrates, both being dependent on the presence of Na(+) ions in the external medium. Interestingly, heteroexchange only occurs in one preferred substrate orientation, i.e. with taurine transported inwards and aspartate outwards, suggesting a mechanism of transinhibition of aspartate uptake by intracellular taurine. Therefore, dEAAT2 is actually an aspartate/taurine transporter. Further studies of this protein are expected to shed light on the role of taurine as a candidate neuromodulator and cell survival factor in the Drosophila nervous system.     

Characterization of the interactions between the glycine transporters GLYT1 and GLYT2 and the SNARE protein syntaxin 1A

In this study we have examined the effect of the SNARE protein syntaxin 1A on the glycine transporters GLYT1 and GLYT2. Our results demonstrate a functional and physical interaction between both glycine transporters and syntaxin 1A. Co-transfection of syntaxin 1A with GLYT1 or GLYT2 in COS cells resulted in approximately 40% inhibition in glycine transport. This inhibition was reversed by the syntaxin 1A-binding protein, Munc18. Furthermore, immunoprecipitation studies showed a physical interaction between syntaxin 1A and both transporters in COS cells and in rat brain tissue. Finally, we conclude that this physical interaction resulted in a partial removal of the glycine transporters from the plasma membrane as demonstrated by biotinylation studies.     

Functional expression of release-regulating glycine transporters GLYT1 on GABAergic neurons and GLYT2 on astrocytes in mouse spinal cord

It is widely accepted that glycine transporters of the GLYT1 type are situated on astrocytes whereas GLYT2 are present on glycinergic neuronal terminals where they mediate glycine uptake. We here used purified preparations of mouse spinal cord nerve terminals (synaptosomes) and of astrocyte-derived subcellular particles (gliosomes) to characterize functionally and morphologically the glial versus neuronal distribution of GLYT1 and GLYT2. Both gliosomes and synaptosomes accumulated [3H]GABA through GAT1 transporters and, when exposed to glycine in superfusion conditions, they released the radioactive amino acid not in a receptor-dependent manner, but as a consequence of glycine penetration through selective transporters. The glycine-evoked release of [3H]GABA was exocytotic from synaptosomes but GAT1 carrier-mediated from gliosomes. Based on the sensitivity of the glycine effects to selective GLYT1 and GLYT2 blockers, the two transporters contributed equally to evoke [3H]GABA release from GABAergic synaptosomes; even more surprising, the 'neuronal' GLYT2 contributed more efficiently than the 'glial' GLYT1 to mediate the glycine effect in [3H]GABA releasing gliosomes. These functional results were largely confirmed by confocal microscopy analysis showing co-expression of GAT1 and GLYT2 in GFAP-positive gliosomes and of GAT1 and GLYT1 in MAP2-positive synaptosomes. To conclude, functional GLYT1 are present on neuronal axon terminals and functional GLYT2 are expressed on astrocytes, indicating not complete selectivity of glycine transporters in their glial versus neuronal localization in the spinal cord.     

The second intracellular loop of the glycine transporter 2 contains crucial residues for glycine transport and phorbol ester-induced regulation

Na+ and Cl(-)-coupled glycine transporters control the availability of glycine neurotransmitter in the synaptic cleft of inhibitory glycinergic pathways. In this report, we have investigated the involvement of the second intracellular loop of the neuronal glycine transporter 2 (GLYT2) on the protein conformational equilibrium and the regulation by 4alpha-phorbol 12 myristate 13-acetate (PMA). By substituting several charged (Lys-415, Lys-418, and Lys-422) and polar (Thr-419 and Ser-420) residues for different amino acids and monitoring plasma membrane expression and kinetic behavior, we found that residue Lys-422 is crucial for glycine transport. The introduction of a negative charge in 422, and to a lower extent in neighboring N-terminal residues, dramatically increases transporter voltage dependence as assessed by response to high potassium depolarizing conditions. In addition, [2-(trimethylammonium)ethyl] methanethiosulfonate accessibility revealed a conformational connection between Lys-422 and the glycine binding/permeation site. Finally, we show that the mutation of positions Thr-419, Ser-420, and mainly Lys-422 to acidic residues abolishes the PMA-induced inhibition of transport activity and the plasma membrane transporter internalization. Our results establish a new structural basis for the action of PMA on GLYT2 and suggest a complex nature of the PMA action on this glycine transporter.     

Expression and transport characterisation of the wheat low-affinity cation transporter (LCT1) in the methylotrophic yeast Pichia pastoris

The low-affinity cation transporter (LCT1) from wheat (Triticum aestivum) was expressed in the methylotrophic yeast Pichia pastoris and its transport characteristics studied employing Ca(45) and Cd(109). A clone (LCT1#3) with the highest uptake of 14pmol of Ca/10(6)cells/10min when exposed to 100microM Ca(45) was chosen for further Ca(45) and Cd(109) transport characteristics. We report for the first time a K(m) for Ca by LCT1 of 0.43+/-0.15mM Ca activity which confirms LCT1 to be a low affinity transporter. Interestingly, the expression of LCT1 in Pichia resulted in reduced Cd(109) uptake compared to wild type cells, when cells were exposed to >or=60microM Cd. This is the first report of the ability of a heterologously expressed transporter to reduce the activity of endogenous transporter proteins to transport Cd. To our knowledge, this is the first demonstration of functional expression of a plant ion transporter using P. pastoris.     

The alanine-serine-cysteine-1 (Asc-1) transporter controls glycine levels in the brain and is required for glycinergic inhibitory transmission

Asc-1 (SLC7A10) is an amino acid transporter whose deletion causes neurological abnormalities and early postnatal death in mice. Using metabolomics and behavioral and electrophysiological methods, we demonstrate that Asc-1 knockout mice display a marked decrease in glycine levels in the brain and spinal cord along with impairment of glycinergic inhibitory transmission, and a hyperekplexia-like phenotype that is rescued by replenishing brain glycine. Asc-1 works as a glycine and L-serine transporter, and its transport activity is required for the subsequent conversion of L-serine into glycine in vivo. Asc-1 is a novel regulator of glycine metabolism and a candidate for hyperekplexia disorders.     

Alkali cation binding and permeation in the rat organic cation transporter rOCT2

Organic cation transporters of the OCT family mediate downhill transport of organic cations, compatible with carrier, pore, or gate-lumen-gate mechanisms. We studied rat OCT2 expressed in Xenopus oocytes by the two-electrode voltage-clamp technique, including membrane capacitance (C(m)) monitoring. Choline, a transported cationic substrate, elicited the expected inward currents but also elicited decreases of C(m). Similar C(m) decreases were caused by the non-transported inhibitors tetrabutylammonium (a cation) and corticosterone (uncharged). Effects on C(m) were voltage-dependent, with a maximum at -140 mV. These findings suggest that the empty rOCT2 protein can undergo an electrogenic conformation change, with one conformation highly favored at physiological voltage. Moreover, alkali cations elicited considerable inward currents and inhibited uptake of [(14)C]tetraethylammonium with a sequence Cs(+) > Rb(+) > K(+) > Na(+) approximately Li(+). Cs(+) affected current and capacitance with similar affinity (K(0.5) approximately 50 mm). Tetraethylammonium inhibited Cs(+) currents in a concentration-dependent manner. Conversely, Cs(+) inhibited tetraethylammonium uptake by a competitive mechanism. Activation energy of the currents estimated from measurements between 12 degrees C and 32 degrees C was approximately 81 kJ/mol for Cs(+) and 39 kJ/mol for tetramethylammonium, compatible with permeation of Cs(+) through rOCT2 along the same path as organic substrates and by a mechanism different from simple electrodiffusion. Rationalization of Cs(+) selectivity in terms of a pore pointed to a pore diameter of approximately 4 A. Intriguingly, that value matches the known selectivity of rOCT2 for organic compounds. Our data show that selective permeability of rOCT2 is not determined by ligand affinity but might rather be understood in terms of the ion channel concept of a distinct "selectivity filter."     

Expression of novel organic cation/carnitine transporter (OCTN2) in the mouse pancreas

Among the organic cation transporters, OCTN2 is identified as the most important carnitine transporter owing to the ability to transport carnitine. Although the OCTN2 is previously found in various tissues, there have been no reports showing the OCTN2 in the pancreas. In this study, we examined the expression and localization of OCTN2 in the mouse pancreas by the aid of an in situ hybridization technique and immunohistochemistry with anti-OCTN2 antibody. As a result, the OCTN2 expression was found in the A-cells for the first time. OCTN2 was not expressed in B-cells, notwithstanding that the metabolism of long-chain fatty acids, which are transported into the mitochondria with the help of carnitine, was expected for fatty acid-stimulated insulin secretion. Thus, this study suggests the possibility of carnitine uptake in the pancreatic A-cells through OCTN2 and implies the presence of carnitine transporter(s) other than OCTN2 in the B-cell.