Chieko Mitsuhata

Molecular cloning and characterization of rat trp homologues from brain

Identification of trp (transient receptor potential) gene from Drosophila photoreceptor and subsequent molecular cloning of the human cDNA homologues suggest its participation in capacitative calcium entry (CCE) or so called store-operated Ca2+ channel (SOC). We identified five different trp-related amplifications of reverse-transcription-polymerase chain reaction (RT-PCR) from rat brain; these corresponded to mouse trp homologues, mtrp1,3,4,5,6 and were distributed in various tissues with multiple expression levels. Two cDNAs, homologous to Drosophila trp from rat brain, designated rtrp3 and rtrp6, were isolated and characterized. By RT-PCR analysis, mRNAs of rtrp3 and rtrp6 were found to be expressed differently in brain and other tissues. In situ hybridization analysis revealed that rtrp6 mRNA was preferentially expressed in hippocampal dentate gyrus and cortical layers II and III. Expression of rat TRP3 and TRP6 in COS cells revealed an increase in CCE, as compared to that in the mock-transfected COS cells of the control. Isolation of cDNAs of rat trp gene family provides a useful model for studying mechanism of CCE.

Dominant Negative Isoform of Rat Norepinephrine Transporter Produced by Alternative RNA Splicing

We have cloned from rat brain a family of alternatively spliced cDNAs from a single gene, which encodes a norepinephrine transporter (NET) having variations at the 3′-region including both coding and noncoding regions. This produces two transporter isoforms, rNETa and rNETb, which differ at their COOH termini. The rNETa isoform reveals a COOH terminus homologous to human NET and transports norepinephrine. In contrast, rNETb revealed no detectable transport function but reduced functional expression of rNETa when both isoforms were expressed in the same cell. Thus, rNETb potentially functions as a dominant negative inhibitor of rNETa activity. Co-expression of rNETb with a γ-aminobutyric acid transporter (rGAT1), a serotonin transporter (rSERT), and a dopamine transporter (rDAT) reduced their transport activity. No reduction was found with the glutamate/aspartate transporter (rGLAST). Alternative RNA splicing of NET suggests a novel mechanism for the regulation of synaptic transmission.

Tyrosine-533 of rat dopamine transporter : involvement in interactions with 1-methyl-4-phenylpyridinium and cocaine

To improve our understanding of structure-function relationships for neurotransmitter transporters, we performed site-directed mutagenesis of the rat dopamine transporter (DAT) and assessed the functions of the mutants in transiently-expressing COS cells. Tyrosine-533 of rat DAT lies in the 11th transmembrane region, where the corresponding amino acid of human DAT is phenylalanine. Alanine substitution of tyrosine-533 (Y533A) conferred an increased affinity for 1-methyl-4-phenylpyridinium (MPP+). Phenylalanine substitution of tyrosine-533 (Y533F) increased the velocity of MPP+ uptake but decreased DATs affinity for MPP+. Cocaines potency in inhibiting dopamine uptake was unchanged with Y533A, but increased with Y533F. Differences in the uptake kinetics and inhibitory potency of cocaine between rat and human DATs were similar to the differences observed between the wild-type and Y533F mutants DATs. Tyrosine-533 may be important for the DAT function and for species differences in transporter functions, including differential sensitivities to cocaine and 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in humans and rats.

MPP+ toxicity and plasma membrane dopamine transporter: study using cell lines expressing the wild-type and mutant rat dopamine transporters.

The Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) causes specific cell death in dopaminergic neurons after accumulation by the dopamine transporter (DAT). COS cells, a non-neuronal cell line insensitive to high doses of MPP+, becomes sensitive to MPP+ when transfected with the rat DAT cDNA. We analyzed the bi-directional transport of MPP+ and its toxicity in several cell lines expressing wild or mutant DATs. Cell death in COS cells expressing wild DAT by exposure to MPP+ was concentration-dependent and cocaine-reversible. Increased wild DAT expression caused higher sensitivities to the toxin in HeLa cells. Although several mutant DATs demonstrated greater transport activity than the wild-type, they displayed similar or lower sensitivity to MPP+ toxicity. Reverse transport of preloaded [3H]MPP+ through DAT was facilitated in COS cells expressing certain mutant DATs, which consistently displayed less sensitivity to MPP+ toxicity. These results suggest that re-distribution of MPP+ due to influx/efflux turnover through the transporter is a key factor in MPP+ toxicity.

Role of C-terminal region in the functional regulation of rat serotonin transporter (SERT).

Previously, we revealed that the state of the actin cytoskeleton affects the uptake activity of the serotonin transporter (SERT). Recently, it was reported that the C-terminus of SERT interacts with MacMARCKS, a substrate of PKC that can bind to the actin cytoskeleton. To elucidate the importance of the C-terminal region in the regulation of SERT activity and the interaction with the actin cytoskeleton, we examined whether the overexpression of the C-terminus affects the transport activity of SERT. To this end, we overexpressed a GFP-fused 30-amino acid construct of the SERT C-terminus (GFP-SERT-CT) in HEK293 cells stably expressing FLAG-tagged SERT (FL-SERT-HEK293 cells). The SERT uptake activity and transporter current were attenuated in GFP-SERT-CT-expressing FL-SERT-HEK293 cells, as compared with GFP-expressing FL-SERT-HEK293 cells. Eadie-Hofstee analysis revealed that GFP-SERT-CT overexpression attenuated the SERT uptake activity by reducing the Vmax, but not changing the Km, which was consistent with the results of experiments on the cell-surface expression of SET using biotinylation/immunoblot analysis. Immunocytochemical analysis demonstrated that GFP-SERT-CT was co-localized with FLAG-SERT and cortical actin at the plasma membrane. In addition, the SERT C-terminus did not affect dopamine transporter activity. These findings showed the significance of the C-terminal region to the functional regulation of SERT, suggesting that GFP-SERT-CT acts as a molecular decoy to disrupt the interaction between SERT and the actin cytoskeleton.

Selective inhibition of monoamine neurotransmitter transporters by synthetic local anesthetics.

Abstract. Synthetic local anesthetics (LAs) have been found to have cocaine-like characteristics with some psychotomimetic action, possibly through monoaminergic neurotransmission. To gain insight into the relation between LA action and monoamine transporters, we investigated the effect of synthetic LAs on neurotransmitter transporters, including monoamine transporters. We used cloned transporter cDNAs and examined transient functional expression in COS cells and stable expression in HeLa cells. Among the LAs tested, procaine and other ester-type LAs inhibited [3H]DA uptake and binding of [3H]2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (CFT), a cocaine analogue, in COS cells expressing rat dopamine transporter (DAT). The inhibition was concentration-dependent. The inhibitory effect on [3H]DA uptake was reversible and not dependent on pH, as observed in HeLa cells stably expressing DAT. Procaine also inhibited uptake of norepinephrine (NE) and serotonin (5-HT) by the norepinephrine transporter (NET) or serotonin transporter (SERT) expressed in COS cells. On the other hand, procaine and other LAs had little or no effect on [3H]GABA and [3H]glutamate uptake in COS cells expressing mouse GABA or rat glutamate/aspartate transporter. IC50 values for [3H]DA uptake inhibition correlated well with those for [3H]CFT binding inhibition, but not with intrinsic anesthetic potency. Kinetic analysis of monoamine uptake inhibition by procaine in COS cells expressing rat DAT, NET or SERT revealed a competitive action similar to that of cocaine. These results demonstrate that certain LAs selectively inhibit monoamine transporters. This might contribute to the cocaine-like psychotomimetic action of certain LAs.

Expression and Function of Variants of Human Catecholamine Transporters Lacking the Fifth Transmembrane Region Encoded by Exon 6

Background The transporters for dopamine (DAT) and norepinephrine (NET) are members of the Na+- and Cl−-dependent neurotransmitter transporter family SLC6. There is a line of evidence that alternative splicing results in several isoforms of neurotransmitter transporters including NET. However, its relevance to the physiology and pathology of the neurotransmitter reuptake system has not been fully elucidated. Methodology/Principal Findings We found novel isoforms of human DAT and NET produced by alternative splicing in human blood cells (DAT) and placenta (NET), both of which lacked the region encoded by exon 6. RT-PCR analyses showed a difference in expression between the full length (FL) and truncated isoforms in the brain and peripheral tissues, suggesting tissue-specific alternative splicing. Heterologous expression of the FL but not truncated isoforms of DAT and NET in COS-7 cells revealed transport activity. However, immunocytochemistry with confocal microscopy and a cell surface biotinylation assay demonstrated that the truncated as well as FL isoform was expressed at least in part in the plasma membrane at the cell surface, although the truncated DAT was distributed to the cell surface slower than FL DAT. A specific antibody to the C-terminus of DAT labeled the variant but not FL DAT, when cells were not treated with Triton for permeabilization, suggesting the C-terminus of the variant to be located extracellulary. Co-expression of the FL isoform with the truncated isoform in COS-7 cells resulted in a reduced uptake of substrates, indicating a dominant negative effect of the variant. Furthermore, an immunoprecipitation assay revealed physical interaction between the FL and truncated isoforms. Conclusions/Significance The unique expression and function and the proposed membrane topology of the variants suggest the importance of isoforms of catecholamine transporters in monoaminergic signaling in the brain and peripheral tissues.

Effectiveness of salivary chromogranin A as a stress index in young children during dental treatment

Abstract It is important to understand the level of stress experienced by children during dental treatment, which can be stressful and even lead to permanent dental phobia. We investigated whether chromogranin A (CgA), a stress marker in adults, is useful for determining stress levels in children. Saliva samples were collected before and after treatment from 5 children (3–5 years) who required treatment more than 5 times in a relatively short period. Their parents completed a questionnaire about their child liking/disliking the clinic visit, how they explained to their children the purpose of the dental visit and the type of treatment sought, and to explain descriptively the difference they noticed in their childs behavior. The CgA levels were significantly higher before than after the treatment in all cases. Pre-treatment CgA values were not always related to dislike for the clinic visit, type of treatment sought, and behavioral reaction to the treatment. The CgA values may have been influenced by the childrens previous experience of the treatment. The results suggest that CgA might be appropriate for verifying childrens stress levels during dental treatment and stress tendency towards dental treatment.