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R P Seal

Publications and source records attributed to R P Seal.

8 recordsLinked to original sources
PubMed2006

The diverse roles of vesicular glutamate transporter 3.

The expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the ability of most traditionally accepted excitatory neurons to release glutamate by exocytosis. However, several cell populations (serotonin and dopamine neurons) have been demonstrated to release glutamate in vitro and do not obviously express these transporters. Rather, these neurons express a novel, third isoform that in fact appears confined to neurons generally associated with a transmitter other than glutamate. They include serotonin and possibly dopamine neurons, cholinergic interneurons in the striatum, and GABAergic interneurons of the hippocampus and cortex. Although the physiological role of VGLUT3 remains largely conjectural, several observations in vivo suggest that the glutamate release mediated by VGLUT3 has an important role in synaptic transmission, plasticity, and development.

Acetylcholine↗
PubMed2001

Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance.

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC(50)s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC(50) for L-glutamate to activate the anion conductance, without affecting the EC(50) for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.

Anions↗
PubMed2001

Effects of threo-beta-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5).

D,L-threo-beta-Benzyloxyaspartate (D,L-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.

Amino Acid Transport System X-AG↗
PubMed2000

A model for the topology of excitatory amino acid transporters determined by the extracellular accessibility of substituted cysteines.

Excitatory amino acid transporters (EAATs) function as both substrate transporters and ligand-gated anion channels. Characterization of the transporter's general topology is the first requisite step in defining the structural bases for these distinct activities. While the first six hydrophobic domains can be readily modeled as conventional transmembrane segments, the organization of the C-terminal hydrophobic domains, which have been implicated in both substrate and ion interactions, has been controversial. Here, we report the results of a comprehensive evaluation of the C-terminal topology of EAAT1 determined by the chemical modification of introduced cysteine residues. Our data support a model in which two membrane-spanning domains flank a central region that is highly accessible to the extracellular milieu and contains at least one reentrant loop domain.

ATP-Binding Cassette Transporters↗
PubMed1999

Excitatory amino acid transporters: a family in flux.

As the most predominant excitatory neurotransmitter, glutamate has the potential to influence the function of most neuronal circuits in the central nervous system. To limit receptor activation during signaling and prevent the overstimulation of glutamate receptors that can trigger excitotoxic mechanisms and cell death, extracellular concentrations of excitatory amino acids are tightly controlled by transport systems on both neurons and glial cells. L-Glutamate is a potent neurotoxin, and the inadequate clearance of excitatory amino acids may contribute to the neurodegeneration seen in a variety of conditions, including epilepsy, ischemia, and amyotrophic lateral sclerosis. To establish the contributions of carrier systems to the etiology of neurological disorders, and to consider their potential utility as therapeutic targets, a detailed understanding of transporter function and pharmacology is required. This review summarizes current knowledge of the structural and functional diversity of excitatory amino acid transporters and explores how they might serve as targets for drug design.

Amino Acid Transport Systems↗
PubMed1998

A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation.

To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.

ATP-Binding Cassette Transporters↗
PubMed1998

Identification and characterization of a cDNA encoding a neuronal glutamate transporter from Drosophila melanogaster.

Sodium-dependent glutamate transporters influence neurotransmission in the central nervous system by removing synaptically released glutamate from the extracellular space and by maintaining extracellular glutamate concentrations below neurotoxic levels. In insects, glutamate also serves as the neurotransmitter at the neuromuscular junction, but the mechanism for neurotransmitter clearance at this synapse has not well-established. Here we report that cloning and characterization of a sodium-dependent glutamate transporter, dEAAT, from Drosophila melanogaster. The 479 amino acid dEAAT gene product is 40-50% homologous to mammalian members of this carrier family. A 3.3 kilobase (kb) transcript for dEAAT was detected in adult fly heads and to a lesser extent in bodies by Northern-blot analysis and was also localized to neurons in the central nervous system by in situ hybridization. The transport activity observed following express of dEAAT in Xenopus oocytes or COS-7 cells shows a high affinity for L-glutamate, L-aspartate and D-aspartate, an absolute dependence on external sodium ions, and considerable stereoselectivity for the transport of L-glutamate over D-glutamate. As has been observed for the human carriers, EAAT 4 and EAAT 5, a significant component of the current activated by L-glutamate application to dEAAT-expressing oocytes appears to arise from the activation of a chloride channel associated with the carrier.

ATP-Binding Cassette Transporters↗