Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT

Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.     

Dynamic equilibrium of neurotransmitter transporters: not just for reuptake anymore

Many electrophysiologists view neurotransmitter transporters as tiny vacuum cleaners, operating continuously to lower extracellular neurotransmitter concentration to zero. However, this is not consistent with their known behavior, instead only reducing extracellular neurotransmitter concentration to a finite, nonzero value at which an equilibrium is reached. In addition, transporters are equally able to go in either the forward or reverse direction, and when they reverse, they release their substrate in a calcium-independent manner. Transporter reversal has long been recognized to occur in response to pathological stimuli, but new data demonstrate that some transporters can also reverse in response to physiologically relevant stimuli. This is consistent with theoretical calculations that indicate that the reversal potentials of GABA and glycine transporters are close to the resting potential of neurons under normal conditions and that the extracellular concentration of GABA is sufficiently high when the GABA transporter is at equilibrium to tonically activate high-affinity extrasynaptic GABAA receptors. The equilibrium for the GABA transporter is not static but instead varies continuously as the driving force for the transporter changes. We propose that the GABA transporter plays a dynamic role in control of brain excitability by modulating the level of tonic inhibition in response to neuronal activity.     

GABA(B) receptors couple to potassium and calcium channels on identified lateral perforant pathway projection neurons

Activation of presynaptic GABA(B) receptors inhibits neurotransmitter release at most cortical synapses, at least in part because of inhibition of voltage-gated calcium channels. One synapse where this is not the case is the lateral perforant pathway synapse onto dentate granule cells in the hippocampus. The current study was conducted to determine whether the neurons that make these synapses express GABA(B) receptors that can couple to ion channels. Perforant pathway projection neurons were labeled by injecting retrograde tracer into the dorsal hippocampus. The GABA(B) receptor agonist baclofen (10 microM) activated inwardly rectifying potassium channels and inhibited currents mediated by voltage-gated calcium channels in retrogradely labeled neurons in layer II of the lateral entorhinal cortex. These effects were reversed by coapplication of the selective GABA(B) receptor antagonist CGP 55845A (1 microM). Equivalent effects were produced by 100 microM adenosine, which inhibits neurotransmitter release at lateral perforant pathway synapses. The effects of baclofen and adenosine on inward currents were largely occlusive. These results suggest that the absence of GABA(B) receptor-mediated presynaptic inhibition at lateral perforant pathway synapses is not simply due to a failure to express these receptors and imply that GABA(B) receptors can either be selectively localized or regulated at terminal versus somatodendritic domains.     

Relations between substrate affinities and charge equilibration rates in the rat GABA cotransporter GAT1

The relations between apparent affinity for substrates and operating rates have been investigated by two-electrode voltage clamp in the GABA transporter rGAT1 expressed in Xenopus oocytes. We have measured the transport current induced by the presence of GABA, as well as the charge equilibration rate in the absence of the neurotransmitter, in various experimental conditions known to affect the transporter characteristics. The apparent affinities for GABA and for Na⁺ were also determined in the same conditions. Two pharmacological actions and three mutated isoforms have been examined. In all cases significant correlations were found between the charge equilibration rates and apparent affinities for both substrates. In particular in the transport process, the apparent affinity for GABA appears to be inversely related to the sum of the unidirectional charge equilibration rates (α+β), while the Na⁺ apparent affinity is directly related to their ratio (β/α). Together these observations suggest a kinetic basis for GABA affinity with higher turnover rates resulting in lower affinity, and indicate that an efficient uptake requires a compromise between these two parameters.     

TRPA1 channels regulate astrocyte resting calcium and inhibitory synapse efficacy through GAT-3

Astrocytes contribute to the formation and function of synapses and are found throughout the brain, where they show intracellular store-mediated Ca(2+) signals. Here, using a membrane-tethered, genetically encoded calcium indicator (Lck-GCaMP3), we report the serendipitous discovery of a new type of Ca(2+) signal in rat hippocampal astrocyte-neuron cocultures. We found that Ca(2+) fluxes mediated by transient receptor potential A1 (TRPA1) channels gave rise to frequent and highly localized 'spotty' Ca(2+) microdomains near the membrane that contributed appreciably to resting Ca(2+) in astrocytes. Mechanistic evaluations in brain slices showed that decreases in astrocyte resting Ca(2+) concentrations mediated by TRPA1 channels decreased interneuron inhibitory synapse efficacy by reducing GABA transport by GAT-3, thus elevating extracellular GABA. Our data show how a transmembrane Ca(2+) source (TRPA1) targets a transporter (GAT-3) in astrocytes to regulate inhibitory synapses.     

Functional expression of γ-amino butyric acid transporter 2 in human and guinea pig airway epithelium and smooth muscle

γ-Amino butyric acid (GABA) is a primary inhibitory neurotransmitter in the central nervous system, and is classically released by fusion of synaptic vesicles with the plasma membrane or by egress via GABA transporters (GATs). Recently, a GABAergic system comprised of GABA(A) and GABA(B) receptors has been identified on airway epithelial and smooth muscle cells that regulate mucus secretion and contractile tone of airway smooth muscle (ASM). In addition, the enzyme that synthesizes GABA, glutamic acid decarboxylase, has been identified in airway epithelial cells; however, the mechanism(s) by which this synthesized GABA is released from epithelial intracellular stores is unknown. We questioned whether any of the four known isoforms of GATs are functionally expressed in ASM or epithelial cells. We detected mRNA and protein expression of GAT2 and -4, and isoforms of glutamic acid decarboxylase in native and cultured human ASM and epithelial cells. In contrast, mRNA encoding vesicular GAT (VGAT), the neuronal GABA transporter, was not detected. Functional inhibition of (3)H-GABA uptake was demonstrated using GAT2 and GAT4/betaine-GABA transporter 1 (BGT1) inhibitors in both human ASM and epithelial cells. These results demonstrate that two isoforms of GATs, but not VGAT, are expressed in both airway epithelial and smooth muscle cells. They also provide a mechanism by which locally synthesized GABA can be released from these cells into the airway to activate GABA(A) channels and GABA(B) receptors, with subsequent autocrine and/or paracrine signaling effects on airway epithelium and ASM.     

Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors

Neurotransmitter transporters can affect neuronal excitability indirectly via modulation of neurotransmitter concentrations or directly via transporter currents. A physiological or pathophysiological role for transporter currents has not been described. We found that GABA transporter 1 (GAT-1) cation currents directly increased GABAergic neuronal excitability and synaptic GABA release in the periaqueductal gray (PAG) during opioid withdrawal in rodents. In contrast, GAT-1 did not indirectly alter GABA receptor responses via modulation of extracellular GABA concentrations. Notably, we found that GAT-1-induced increases in GABAergic activity contributed to many PAG-mediated signs of opioid withdrawal. Together, these data support the hypothesis that GAT-1 activity directly produces opioid withdrawal signs through direct hyperexcitation of GABAergic PAG neurons and nerve terminals, which presumably enhances GABAergic inhibition of PAG output neurons. These data provide, to the best of our knowledge, the first evidence that dysregulation of a neurotransmitter transporter current is important for the maladaptive plasticity that underlies opiate withdrawal.     

The ABC-transporter signature motif is required for peptide translocation but not peptide binding by TAP

The transporter associated with antigen processing (TAP) translocates peptides from their site of generation in the cytosol to the lumen of the endoplasmic reticulum for binding to MHC class I molecules. TAP is a member of the ATP-binding cassette (ABC) transporter family whose members utilize energy from ATP hydrolysis to translocate substrates across membranes. The highly conserved nucleotide-binding domains of ABC transporters couple ATP hydrolysis to substrate translocation by the membrane domains. The conserved 'signature motif' can be identified in the nucleotide-binding domains of all ABC transporters, and may play a role in ATP hydrolysis. Here we show that introduction of mutations into the signature motifs of either TAP1 or TAP2 inhibits the translocation of peptide without affecting binding of either peptide or ATP by TAP. We therefore conclude that the signature motifs in both TAP1 and TAP2 are required after peptide binding to facilitate peptide translocation by TAP.     

Reversal or reduction of glutamate and GABA transport in CNS pathology and therapy

A dysfunction of amino acid neurotransmitter transporters occurs in a number of central nervous system disorders, including stroke, epilepsy, cerebral palsy and amyotrophic lateral sclerosis. This dysfunction can comprise a reversal of transport direction, leading to the release of neurotransmitter into the extracellular space, or an alteration in transporter expression level. This review analyses the role of glutamate and GABA transporters in the pathogenesis and therapy of a number of acute and chronic neurological disorders.     

Stable expression of the vesicular GABA transporter following photothrombotic infarct in rat brain

Before exocytotic release of the inhibitory neurotransmitter GABA, this amino acid has to be stored in synaptic vesicles. Accumulation of GABA in vesicles is achieved by a specific membrane-integrated transporter termed vesicular GABA transporter. This vesicular protein is mainly located at presynaptic terminals of GABAergic interneurons. In the present study we investigated the effects of focal ischemia on the expression of the vesicular GABA transporter. Vesicular GABA transporter mRNA and protein expression was examined after photothrombosis in different cortical and hippocampal brain regions of Wistar rats. In situ hybridization and quantitative real-time RT-PCR were performed to analyze vesicular GABA transporter mRNA. Both vesicular GABA transporter mRNA-stained perikarya and mRNA expression levels remained unaffected. Vesicular GABA transporter protein-containing synaptic terminals and somata were visualized by immunohistochemistry. The pattern of vesicular GABA transporter immunoreactivity as well as the protein expression level revealed by semiquantitative image analysis and by Western blot remained stable after stroke. The steady expression of vesicular GABA transporter mRNA and protein after photothrombosis indicates that the exocytotic release mechanism of GABA is not affected by ischemia.     

Glycerol-3-phosphate transporter of Escherichia coli: structure, function and regulation

Glycerol-3-phosphate (G3P) plays a major role in glycolysis and phospholipid biosynthesis in the cell. Escherichia coli uses a secondary membrane transporter protein, GlpT, to uptake G3P into the cytoplasm. The crystal structure of the protein was recently determined to 3.3 A resolution. The protein consists of an N- and a C-terminal domain, each formed by a compact bundle of six transmembrane alpha-helices. The substrate-translocation pore is found at the domain interface and faces the cytoplasm. At the closed end of the pore is the substrate binding site, which is formed by two arginine residues. In combination with biochemical data, the crystal structure suggests a single binding site, alternating access mechanism for substrate translocation, namely, the substrate bound at the N- and C-terminal domain interface is transported across the membrane via a rocker-switch type of movement of the domains. Furthermore, GlpT may serve as a structural and mechanistic paradigm for other secondary active membrane transporters.     

Steady-state kinetic characterization of the mouse B0AT1 sodium-dependent neutral amino acid transporter

The members of the neurotransmitter transporter family SLC6A exhibit a high degree of structural homology; however differences arise in many aspects of their transport mechanisms. In this study we report that mouse B(0)AT1 (mouse Slc6a19) mediates the electrogenic transport of a broad range of neutral amino acids but not of the chemically similar substrates transported by other SLC6A family members. Cotransport of L: -Leu and Na(+) generates a saturable, reversible, inward current with Michaelis-Menten kinetics (Hill coefficient approximately 1) yielding a K(0.5) for L: -Leu of 1.16 mM and for Na(+) of 16 mM at a holding potential of -50 mV. Changing the membrane voltage influences both substrate binding and substrate translocation. Li(+) can substitute partially for Na(+) in the generation of L: -Leu-evoked inward currents, whereas both Cl(-) and H(+) concentrations influence its magnitude. The simultaneous measurement of charge translocation and L: -Leu uptake in the same cell indicates that B(0)AT1 transports one Na(+) per neutral amino acid. This appears to be accomplished by an ordered, simultaneous mechanism, with the amino acid binding prior to the Na(+), followed by the simultaneous translocation of both co-substrates across the plasma membrane. From this kinetic analysis, we conclude that the relatively constant [Na(+)] along the renal proximal tubule both drives the uptake of neutral amino acids via B(0)AT1 thermodynamically and ensures that, upon binding, these are translocated efficiently into the cell.     

Molecular mechanisms of pulmonary peptidomimetic drug and peptide transport

The aerosolic administration of peptidomimetic drugs could play a major role in the future treatment of various pulmonary and systemic diseases, because rational drug design offers the potential to specifically generate compounds that are transported efficiently into the epithelium by distinct carrier proteins such as the peptide transporters. From the two presently known peptide transporters, PEPT1 and PEPT2, which have been cloned from human tissues, the high-affinity transporter PEPT2 is expressed in the respiratory tract epithelium. The transporter is an integral membrane protein with 12 membrane-spanning domains and mediates electrogenic uphill peptide and peptidomimetic drug transport by coupling of substrate translocation to a transmembrane electrochemical proton gradient serving as driving force. In human airways, PEPT2 is localized to bronchial epithelium and alveolar type II pneumocytes, and transport studies revealed that both peptides and peptidomimetic drugs such as antibiotic, antiviral, and antineoplastic drugs are carried by the system. PEPT2 is also responsible for the transport of delta-aminolevulinic acid, which is used for photodynamic therapy and the diagnostics of pulmonary neoplasms. Based on the recent progress in understanding the structural requirements for substrate binding and transport, PEPT2 becomes a target for a rational drug design that may lead to a new generation of respiratory drugs and prodrugs that can be delivered to the airways via the peptide transporter.     

Ca(2+) regulation of the carrier-mediated gamma-aminobutyric acid release from isolated synaptic plasma membrane vesicles

The regulation of the carrier-mediated gamma-aminobutyric acid (GABA) efflux was studied in isolated synaptic plasma membrane (SPM) vesicles, which are particularly useful to study neurotransmitter release without interference of the exocytotic machinery. We investigated the effect of micromolar intravesicular Ca(2+) on the GABA release from SPM vesicles under conditions of basal release (superfusion with 150 mM NaCl), homoexchange (superfusion with 500 microM GABA) and K(+) depolarization-induced release (superfusion with 150 mM KCl). We observed that, in the presence of intravesicular Ca(2+) (10 microM), the maximal velocity (J(max)) of K(+) depolarization-induced GABA release is decreased by about 64%, and this effect was abolished in the presence of the channel blocker, La(3+). In contrast, the other mechanisms were not significantly altered by these cations. In agreement with our earlier results, inhibition of GABA uptake by intravesicular Ca(2+) was also observed by determining the kinetic parameters (K(0.5) and J(max)) of influx into the SPM vesicles. Under these conditions, the J(max) of GABA uptake was 17.4 pmol/s per mg protein, whereas in control experiments (absence of Ca(2+)), this value achieved 25.5 pmol/s per mg protein. The inhibitory effect of Ca(2+) on translocation of GABA across SPM appears to be mediated by calcium/calmodulin activation of protein phosphatase 2B (calcineurin), since it was completely relieved by W7 (calmodulin antagonist) and by cyclosporin A (calcineurin inhibitor). These results show that the GABA transport system, operating either in forward or backward directions, requires phosphorylation of internally localized calcineurin-sensitive sites to achieve maximal net translocation velocity.     

Selective inhibition of spontaneous but not Ca2+ -dependent release machinery by presynaptic group II mGluRs in rat cerebellar slices

Two main forms of neurotransmitter release are known: action potential-evoked and spontaneous release. Action potential-evoked release depends on Ca2+ entry through voltage-gated Ca2+ channels, whereas spontaneous release is thought to be Ca2+ -independent. Generally, spontaneous and action potential-evoked release are believed to use the same release machinery to release neurotransmitter. This study shows, using the whole cell patch-clamp technique in rat cerebellar slices, that at the interneuron- Purkinje cell synapse activation of presynaptic group II metabotropic glutamate receptors suppresses spontaneous GABA release through a mechanism independent of voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Ca2+ release from intracellular Ca2+ stores, suggesting that the metabotropic receptors target the release machinery directly. Voltage gated Ca2+ channel-independent release following increased presynaptic cAMP production is similarly inhibited by these metabotropic receptors. In contrast, both voltage-gated Ca2+ channel-dependent and presynaptic N-methyl-D-aspartate receptor-dependent GABA release were unaffected by activation of group II metabotropic glutamate receptors. Hence, the mechanisms underlying spontaneous and Ca2+ -dependent GABA release are distinct in that only the former is blocked by group II metabotropic glutamate receptors. Thus the same neurotransmitter, glutamate, can activate or inhibit neurotransmitter release by selecting different receptors that target different release machineries.     

Calcium influx through N-methyl-D-aspartate receptors triggers GABA release at interneuron-Purkinje cell synapse in rat cerebellum

Ca(2+)-dependent neurotransmitter release was originally thought to occur only following activation of presynaptic voltage-gated calcium channels after a presynaptic action potential. Recent evidence suggests that not only opening of voltage-gated but also ligand-gated ion channels, such as neurotransmitter receptors, can trigger exocytosis, as well as Ca(2+) release from intracellular Ca(2+) stores. It was shown that activation of N-methyl-d-aspartate (NMDA) receptors on presynaptic interneurons led to increases in GABA release from these neurons onto postsynaptic Purkinje cells in rat cerebellum in the presence of tetrodotoxin (TTX), suggesting a presynaptic location for the underlying NMDA receptors. However, the mechanism for the NMDA-induced increase in GABA release remained unclear. The present study addresses the question whether Ca(2+) influx through presynaptic NMDA receptors alone is sufficient to trigger presynaptic GABA release at this synapse or whether activation of presynaptic NMDA receptors leads to opening of voltage-gated Ca(2+) channels, thereby increasing exocytosis. The results suggest that the NMDA-induced increase in presynaptic GABA release neither requires activation of presynaptic voltage-gated Ca(2+) channels nor Ca(2+) release from presynaptic Ca(2+) stores. It is concluded that Ca(2+) influx through the NMDA receptor alone is sufficient to drive presynaptic GABA release at the rat interneuron-Purkinje cell synapse.     

Clustered K channel complexes in axons

Voltage-gated K⁺ (Kv) channels regulate diverse neuronal properties including action potential threshold, amplitude, and duration, frequency of firing, neurotransmitter release, and resting membrane potential. In axons, Kv channels are clustered at a variety of functionally important sites including axon initial segments, juxtaparanodes of myelinated axons, nodes of Ranvier, and cerebellar basket cell terminals. These channels are part of larger protein complexes that include cell adhesion molecules and scaffolding proteins. These interacting proteins play important roles in recruiting K⁺ channels to distinct axonal domains. Here, I review the composition, functions, and mechanism of localization of these K⁺ channel complexes in axons.     

Two sites of action for synapsin domain E in regulating neurotransmitter release

Synapsins, a family of synaptic vesicle proteins, have been shown to regulate neurotransmitter release; the mechanism(s) by which they act are not fully understood. Here we have studied the role of domain E of synapsins in neurotransmitter release at the squid giant synapse. Two squid synapsin isoforms were cloned and found to contain a carboxy (C)-terminal domain homologous to domain E of the vertebrate a-type synapsin isoforms. Presynaptic injection of a peptide fragment of domain E greatly reduced the number of synaptic vesicles in the periphery of the active zone, and increased the rate and extent of synaptic depression, suggesting that domain E is essential for synapsins to regulate a reserve pool of synaptic vesicles. Domain E peptide had no effect on the number of docked synaptic vesicles, yet reversibly inhibited and slowed the kinetics of neurotransmitter release, indicating a second role for synapsins that is more intimately associated with the release process itself. Thus, synapsin domain E is involved in at least two distinct reactions that are crucial for exocytosis in presynaptic terminals.     

Molecular regulation of glutamate and GABA transporter proteins by valproic acid in rat hippocampus during epileptogenesis

Epileptiform discharges and behavioral seizures may be the consequences of the presence of either excessive excitation associated with the neurotransmitter glutamate or from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). Synaptic effects of these neurotransmitters are terminated by the action of transporter proteins that remove these amino acids from the synaptic cleft. The glial transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), and the neuronal transporter excitatory amino acids carrier-1 (EAAC-1) limit excitation initiated by synaptic release of glutamate. Transporter proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3) remove GABA from synaptic regions. To assess the molecular effects of the antiepileptic drug valproate, albino rats with chronic, spontaneous, recurrent seizures induced by amygdalar injection of FeCl3 were treated for 14 days with either valproic acid or with saline as an injection control. Regions of the hippocampus were assayed for glutamate and GABA transporters by western blot. While epileptogenesis is thought to correlate with the downregulation of GLAST and upregulation of EAAC-1, valproate caused an increase in the quantity of GLAST protein measured in the hippocampus. Valproate treatment decreased GLT-1 in both control and experimental animals in both hippocampi. EAAC-1 was unchanged by valproate treatment. GABA transporters GAT-1 and GAT-3 in the hippocampus were upregulated by FeCl3 injection into the amygdala. However, valproate caused the downregulation of these GABA transporters in both control and experimental animals. Altered molecular regulation of glutamate appears to be critical in the development of sustained, spontaneous limbic seizures. Our data suggest that valproate may have unique mechanisms of action; specifically, it may affect the removal of glutamate by upregulating GLAST and decreasing GABA transport, which could result in increased tissue concentrations of GABA.