The SLC36 family: proton-coupled transporters for the absorption of selected amino acids from extracellular and intracellular proteolysis

Whilst Na(+) has replaced H(+) as a major transport driving force at the plasma membrane of animal cells, the evolutionarily older H(+)-driven systems persist on endomembranes and at the plasma membrane of specialized cells. The first member of the SLC36 family, present in both intracellular and plasma membranes, was identified independently as a lysosomal amino acid transporter (LYAAT1) responsible for the export of lysosomal proteolysis products into the cytosol and as a proton/amino acid transporter (PAT1) responsible for the absorption of amino acids in the gut. In addition to LYAAT1/PAT1, the family comprises another characterized member, PAT2, and two orphan transporters. Both PAT1 and PAT2 mediate 1:1 symport of protons and small neutral amino acids such as glycine, alanine, and proline. Their mRNAs are broadly and differentially expressed in mammalian tissues. The PAT1 protein localizes to lysosomes in brain neurons, but is also found in the apical membrane of intestinal epithelial cells with a role in the absorption of amino acids from luminal protein digestion. In both cases, protons supplied by the lysosomal H(+)-ATPase or by the acidic microclimate of the brush border membrane drive transport of the amino acids into the cytosol. The subcellular localization and physiological role of PAT2 have still to be determined. SLC36 transporters are related distantly to other proton-coupled amino acid transporters, such as the vesicular neurotransmitter transporter VIAAT/VGAT (SLC32) and system N transporters (SLC38 family).     

Amino acid transporter (VIAAT,VGLUT2) and chloride cotransporter (KCC1, KCC2 and NKCC1) expression in the vestibular nuclei of intact and labyrinthectomized rat

We report the first investigation of whether unilateral labyrinthectomy in adult rats affects the expression of two amino acid transporters, vesicular glutamate transporter 2 (VGLUT2) and vesicular inhibitory amino acid transporter (VIAAT) and of chloride cotransporters (KCC1, KCC2 and NKCC1) in the intact and deafferented medial vestibular nuclei (MVN). In situ hybridization with specific radioactive oligonucleotide probes and immunofluorescent methods were used in normal and unilaterally labyrinthectomized rats at various times following the lesion: 5 h, and 1, 3 and 8 days. In normal animals, several brainstem regions including the lateral, medial, superior and inferior vestibular nuclei contained VGLUT2, VIAAT and KCC2 mRNA. In contrast, no or a very faint labeling was observed with KCC1 and NKCC1 probes. In unilaterally lesioned rats, there was no asymmetry between the two MVN with any of the oligonucleotide probes at any time after the lesion. Similarly, there were no differences in the intensity of MVN labeling between controls and lesioned animals. Finally, no over-expression of the cotransporter KCC1 and NKCC1 was found in ipsilateral or controlateral MVN in lesioned rats at any time. Immunohistochemical experiments gave similar conclusions. Our findings suggest that the recovery of the resting discharge of the deafferented MVN neurons, and consequently the functional compensation of the deficits, are not dependent on changes in the expression of amino acid transporters (VIAAT, VGLUT2), and chloride cotransporters (KCC1, KCC2 and NKCC1) or on their mRNAs.     

Vesicular inhibitory amino acid transporter is expressed in gamma-aminobutyric acid (GABA)-containing astrocytes in rat pineal glands

Gamma-aminobutyric acid (GABA) is an inhibitory amino acid and acts as an intercellular transmitter in the central nervous system and peripheral tissues. In pineal glands, GABA is supposed to be a paracrine-like modulator of secretion of melatonin, although its mode of action, especially the sites of GABA signal appearance, is unknown. Vesicular inhibitory amino acid transporter (VIAAT) is a potential marker for the GABAergic phenotype. Here we presented evidence that VIAAT is expressed in GFAP-expressing astrocytes and a subpopulation of OX42-expressing microglia, but not in pinealocytes in cultured cells of rat pineal glands. The VIAAT-expressing cells also exhibit GABA immunoreactivity. Essentially the same results were obtained for pineal glands. These results suggest that GABA is stored and secreted from astrocytes and a subpopulation of microglia in pineal glands.     

H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine

The H(+)-electrochemical gradient was originally considered as a driving force for solute transport only across cellular membranes of bacteria, plants and yeast. However, in the mammalian small intestine, a H(+)-electrochemical gradient is present at the epithelial brush-border membrane in the form of an acid microclimate. Over recent years, a large number of H(+)-coupled cotransport mechanisms have been identified at the luminal membrane of the mammalian small intestine. These transporters are responsible for the initial stage in absorption of a remarkable variety of essential and non-essential nutrients and micronutrients, including protein digestion products (di/tripeptides and amino acids), vitamins, short-chain fatty acids and divalent metal ions. Proton-coupled cotransporters expressed at the mammalian small intestinal brush-border membrane include: the di/tripeptide transporter PepT1 (SLC15A1); the proton-coupled amino-acid transporter PAT1 (SLC36A1); the divalent metal transporter DMT1 (SLC11A2); the organic anion transporting polypeptide OATP2B1 (SLC02B1); the monocarboxylate transporter MCT1 (SLC16A1); the proton-coupled folate transporter PCFT (SLC46A1); the sodium-glucose linked cotransporter SGLT1 (SLC5A1); and the excitatory amino acid carrier EAAC1 (SLC1A1). Emerging research demonstrates that the optimal intestinal absorptive capacity of certain H(+)-coupled cotransporters (PepT1 and PAT1) is dependent upon function of the brush-border Na(+)-H(+) exchanger NHE3 (SLC9A3). The high oral bioavailability of a large number of pharmaceutical compounds results, in part, from absorptive transport via the same H(+)-coupled cotransporters. Drugs undergoing H(+)-coupled cotransport across the intestinal brush-border membrane include those used to treat bacterial infections, hypercholesterolaemia, hypertension, hyperglycaemia, viral infections, allergies, epilepsy, schizophrenia, rheumatoid arthritis and cancer.     

Mammalian ion-coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy-coupling mechanisms. Ion-coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co-transport of H+, Na+, Cl- and/or to the countertransport of K+ or OH-. By contrast, ATP-dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion-coupled transporter cDNAs and revealed new insights into structural designs, energy-coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion-coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co-transporters SGLT1 and SGLT2, the H(+)-coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)- and K(+)-dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion-coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)-coupled transporter family which includes the Na+/glucose co-transporters SGLT1, SGLT2, SGLT3 (SAAT-pSGLT2) and the inositol transporter SMIT, (2) the Na(+)- and Cl(-)-coupled transporter family which includes the neurotransmitter transporters of gamma-amino-butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta-amino acids, (3) the Na(+)- and K(+)-dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT-1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)-coupled oligopeptide transporter family which includes the intestinal H(+)-dependent oligopeptide transporter PepT1.     

The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects

The solute carrier family 1 (SLC1) includes five high-affinity glutamate transporters, EAAC1, GLT-1, GLAST, EAAT4 and EAAT5 (SLC1A1, SLC1A2, SLC1A3, SLC1A6, and SLC1A7, respectively) as well as the two neutral amino acid transporters, ASCT1 and ASCT2 (SLC1A4 and ALC1A5, respectively). Although each of these transporters have similar predicted structures, they exhibit distinct functional properties which are variations of a common transport mechanism. The high-affinity glutamate transporters mediate transport of l-Glu, l-Asp and d-Asp, accompanied by the cotransport of 3 Na(+) and 1 H(+), and the countertransport of 1 K(+), whereas ASC transporters mediate Na(+)-dependent exchange of small neutral amino acids such as Ala, Ser, Cys and Thr. The unique coupling of the glutamate transporters allows uphill transport of glutamate into cells against a concentration gradient. This feature plays a crucial role in protecting neurons against glutamate excitotoxicity in the central nervous system. During pathological conditions, such as brain ischemia (e.g. after a stroke), however, glutamate exit can occur due to "reversed glutamate transport", which is caused by a reversal of the electrochemical gradients of the coupling ions. Selective inhibition of the neuronal glutamate transporter EAAC1 (SLC1A1) may be of therapeutic interest to block glutamate release from neurons during ischemia. On the other hand, upregulation of the glial glutamate transporter GLT1 (SLC1A2) may help protect motor neurons in patients with amyotrophic lateral sclerosis (ALS), since loss of function of GLT1 has been associated with the pathogenesis of certain forms of ALS.     

Evidence for microvesicular storage and release of glycine in rodent pinealocytes

Prompted by previous studies suggesting a regulatory role for the inhibitory amino acid gamma-aminobutyric acid (GABA) within the mammalian pineal gland, we carried out a study of rat and gerbil pineal organs to elucidate whether there is evidence for a vesicular storage and release of GABA and/or glycine. Immunohistochemistry revealed the presence of the vesicular inhibitory amino acid transporter in pinealocytes. Moreover, we found that, in addition to glutamate and aspartate, cultured pinealocytes also released glycine upon stimulation by depolarizing concentrations of KCl, whereas the content of GABA in the culture medium did not exceed the detection limit either under control conditions or following KCl application. Therefore, we propose that glycine is a further component of the paracrine signaling system within the pineal organ which is based on the compartment of synaptic-like microvesicles (SLMVs) inside pinealocytes.     

Inhibitory postsynaptic membrane specializations are formed in gephyrin-deficient mice

Gephyrin is a major postsynaptic scaffolding protein at GABAergic and glycinergic inhibitory synapses. Gephyrin-deficient (geph^−/−) mice die after birth due to disinhibition of motor and sensory pathways resulting from a lack of postsynaptic glycine receptor and GABA_A receptor clusters. Here, immunoelectron and confocal microscopy revealed that postsynaptic membrane specializations are formed in the absence of gephyrin. First, in brainstem sections obtained from newborn geph^−/− mice inhibitory nerve terminals identified by immunogold labeling of either the vesicular inhibitory amino acid transporter (VIAAT) or GABA were found to be apposed to postsynaptic membrane areas decorated by electron-dense material. Second, neuroligin-2, a membrane protein of inhibitory postsynapses, was clustered beneath glutamate decarboxylase 65 (GAD-65) positive nerve terminals in geph^−/− hippocampal cultures. These results indicate that proteins other than gephyrin define the ultrastructure of inhibitory postsynaptic membrane specializations.     

Localization of the Na(+)-coupled neutral amino acid transporter 2 in the cerebral cortex

We studied the distribution and cellular localization of Na(+)-coupled neutral amino acid transporter 2, a member of the system A family of amino acid transporters, in the rat and human cerebral cortex using immunocytochemical methods. Na(+)-coupled neutral amino acid transporter 2-positive neurons were pyramidal and non-pyramidal, and Na(+)-coupled neutral amino acid transporter 2/GABA double-labeling studies revealed that Na(+)-coupled neutral amino acid transporter 2 was highly expressed by GABAergic neurons. Double-labeling studies with the synaptophysin indicated that rare axon terminals express Na(+)-coupled neutral amino acid transporter 2. Na(+)-coupled neutral amino acid transporter 2-immunoreactivity was also found in astrocytes, leptomeninges, ependymal cells and choroid plexus. Electron microscopy showed robust Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in the somato-dendritic compartment of neurons and in glial processes, but, as in the case of double-labeling studies, failed to reveal Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in terminals. To rule out the possibility that the absence of Na(+)-coupled neutral amino acid transporter 1- and Na(+)-coupled neutral amino acid transporter 2-positive terminals was due to insufficient antigen detection, we evaluated Na(+)-coupled neutral amino acid transporter 1/synaptophysin and Na(+)-coupled neutral amino acid transporter 2/synaptophysin coexpression using non-standard immunocytochemical procedures and found that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2+ terminals were rare in all conditions. These findings indicate that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2 are virtually absent in cortical terminals, and suggest that they do not contribute significantly to replenishing the Glu and GABA transmitter pools through the glutamate-glutamine cycle. The strong expression of Na(+)-coupled neutral amino acid transporter 2 in the somato-dendritic compartment and in non-neuronal elements that are integral parts of the blood-brain and brain-cerebrospinal fluid barrier suggests that Na(+)-coupled neutral amino acid transporter 2 plays a role in regulating the levels of Gln and other amino acids in the metabolic compartment of cortical neurons.     

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.     

The molecular basis of neutral aminoacidurias

Recent success in the molecular cloning and identification of apical neutral amino acid transporters has shed a new light on inherited neutral amino acidurias, such as Hartnup disorder and Iminoglycinuria. Hartnup disorder is caused by mutations in the neutral amino acid transporter B(0) AT1 (SLC6A19). The transporter is found in kidney and intestine, where it is involved in the resorption of all neutral amino acids. The molecular defect underlying Iminoglycinuria has not yet been identified. However, two transporters, the proton amino acid transporter PAT1 (SLC36A1) and the IMINO transporter (SLC6A20) appear to play key roles in the resorption of glycine and proline. A model is presented, involving all three transporters that can explain the phenotypic variability of iminoglycinuria.     

Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6

The SLC6 family is a diverse set of transporters that mediate solute translocation across cell plasma membranes by coupling solute transport to the cotransport of sodium and chloride down their electrochemical gradients. These transporters probably have 12 transmembrane domains, with cytoplasmic N- and C-terminal tails, and at least some may function as homo-oligomers. Family members include the transporters for the inhibitory neurotransmitters GABA and glycine, the aminergic transmitters norepinephrine, serotonin, and dopamine, the osmolytes betaine and taurine, the amino acid proline, and the metabolic compound creatine. In addition, this family includes a system B(0+) cationic and neutral amino acid transporter, and two transporters for which the solutes are unknown. In general, SLC6 transporters act to regulate the level of extracellular solute concentrations. In the central and the peripheral nervous system, these transporters can regulate signaling among neurons, are the sites of action of various drugs of abuse, and naturally occurring mutations in several of these proteins are associated with a variety of neurological disorders. For example, transgenic animals lacking specific aminergic transporters show profoundly disturbed behavioral phenotypes and probably represent excellent systems for investigating psychiatric disease. SLC6 transporters are also found in many non-neural tissues, including kidney, intestine, and testis, consistent with their diverse physiological roles. Transporters in this family represent attractive therapeutic targets because they are subject to multiple forms of regulation by many different signaling cascades, and because a number of pharmacological agents have been identified that act specifically on these proteins.     

The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus

The extracellular space of the brain contains γ-aminobutyric acid (GABA) that activates extrasynaptic GABA_A receptors mediating tonic inhibition. The source of this GABA is uncertain: it could be overspill of vesicular release, non-vesicular leakage, reverse transport, dying cells or glia. Using a novel approach, we simultaneously measured phasic and tonic inhibitory currents and assessed their correlation. Enhancing or diminishing vesicular GABA release in hippocampal neurons caused highly correlated changes in the two inhibitions. During high-frequency phasic inhibitory bursts, tonic current was also enhanced as shown by simulating the summation of IPSCs and by recordings in knockout mice devoid of tonic inhibitory current. When vesicular release was reduced by blocking action potentials or the vesicular GABA transporter, phasic and tonic currents decreased in a correlated fashion. Our results are consistent with most of hippocampal tonic inhibitory current being mediated by GABA released from the very vesicles responsible for activating phasic inhibition.     

GABA(B) receptors at glutamatergic synapses in the rat striatum

Although multiple effects of GABA(B) receptor activation on synaptic transmission in the striatum have been described, the precise locations of the receptors mediating these effects have not been determined. To address this issue, we carried out pre-embedding immunogold electron microscopy in the rat using antibodies against the GABA(B) receptor subunits, GABA(B1) and GABA(B2). In addition, to investigate the relationship between GABA(B) receptors and glutamatergic striatal afferents, we used antibodies against the vesicular glutamate transporters, vesicular glutamate transporter 1 and vesicular glutamate transporter 2, as markers for glutamatergic terminals. Immunolabeling for GABA(B1) and GABA(B2) was widely and similarly distributed in the striatum, with immunogold particles localized at both presynaptic and postsynaptic sites. The most commonly labeled structures were dendritic shafts and spines, as well as terminals forming asymmetric and symmetric synapses. In postsynaptic structures, the majority of labeling associated with the plasma membrane was localized at extrasynaptic sites, although immunogold particles were also found at the postsynaptic specialization of some symmetric, putative GABAergic synapses. Labeling in axon terminals was located within, or at the edge of, the presynaptic active zone, as well as at extrasynaptic sites. Double labeling for GABA(B) receptor subunits and vesicular glutamate transporters revealed that labeling for both GABA(B1) and GABA(B2) was localized on glutamatergic axon terminals that expressed either vesicular glutamate transporter 1 or vesicular glutamate transporter 2. The patterns of innervation of striatal neurons by the vesicular glutamate transporter 1- and vesicular glutamate transporter 2-positive terminals suggest that they are selective markers of corticostriatal and thalamostriatal afferents, respectively. These results thus provide evidence that presynaptic GABA(B) heteroreceptors are in a position to modulate the two major excitatory inputs to striatal spiny projection neurons arising in the cortex and thalamus. In addition, presynaptic GABA(B) autoreceptors are present on the terminals of spiny projection neurons and/or striatal GABAergic interneurons. Furthermore, the data indicate that GABA may also affect the excitability of striatal neurons via postsynaptic GABA(B) receptors.     

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.     

Kidney amino acid transport

Near complete reabsorption of filtered amino acids is a main specialized transport function of the kidney proximal tubule. This evolutionary conserved task is carried out by a subset of luminal and basolateral transporters that together form the transcellular amino acid transport machinery similar to that of small intestine. A number of other amino acid transporters expressed in the basolateral membrane of proximal kidney tubule cells subserve either specialized metabolic functions, such as the production of ammonium, or are part of the cellular housekeeping equipment. A new finding is that the luminal Na⁺-dependent neutral amino acid transporters of the SLC6 family require an associated protein for their surface expression as shown for the Hartnup transporter B⁰AT1 (SLC6A19) and suggested for the l-proline transporter SIT1 (IMINO^B, SLC6A20) and for B⁰AT3 (XT2, SLC6A18). This accessory subunit called collectrin (TMEM27) is homologous to the transmembrane anchor region of the renin–angiotensin system enzyme ACE2 that we have shown to function in small intestine as associated subunit of the luminal SLC6 transporters B⁰AT1 and SIT1. Some mutations of B⁰AT1 differentially interact with these accessory subunits, providing an explanation for differential intestinal phenotypes among Hartnup patients. The basolateral efflux of numerous amino acids from kidney tubular cells is mediated by heteromeric amino acid transporters that function as obligatory exchangers. Thus, other transporters within the same membrane need to mediate the net efflux of exchange substrates, controlling thereby the net basolateral amino transport and thus the intracellular amino acid concentration.     

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.     

The vesicular amine transporter family (SLC18): amine/proton antiporters required for vesicular accumulation and regulated exocytotic secretion of monoamines and acetylcholine

The vesicular amine transporters (VATs) are expressed as integral proteins of the lipid bilayer membrane of secretory vesicles in neuronal and endocrine cells. Their function is to allow the transport of acetylcholine (by the vesicular acetylcholine transporter VAChT; SLC18A3) and biogenic amines (by the vesicular monoamine transporters VMAT1 and VMAT2; SLC18A1 and SLC18A2) into secretory vesicles, which then discharge them into the extracellular space by exocytosis. Transport of positively charged amines by members of the SLC18 family in all cases utilizes an electrochemical gradient across the vesicular membrane established by proton pumping into the vesicle via a vacuolar ATPase; the amine is accumulated in the vesicle at the expense of the proton gradient, at a ratio of one translocated amine per two translocated protons. The members of the SLC18 family have become important histochemical markers for chemical coding in neuroendocrine tissues and cells. The structural basis of their remarkable ability to transport positively charged amines against a very large concentration gradient, as well as potential disease association with impaired transporter function and expression, are under intense investigation.     

Effect of diazoxide on regulation of vesicular and plasma membrane GABA transporter genes and proteins in hippocampus of rats subjected to picrotoxin-induced kindling

Epileptiform discharges and behavioral seizures may be the consequences of excess excitation from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). GABA is taken up and accumulated in synaptic vesicles by the action of vesicular GABA transporter (VGAT) before its release into the synaptic cleft, and removed from synaptic regions by the action of transporter proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3). In this experiment, the effects of diazoxide (DIZ) on the VGAT, GAT-1 and GAT-3 mRNA and protein levels in hippocampus, and on the seizure activities of picrotoxin (PTX)-induced kindling rats were observed. DIZ caused increase in the quantity of VGAT mRNAs and proteins, and down regulation of GABA transporters GAT-1 and GAT-3 mRNAs and proteins after the PTX re-kindling. Furthermore, DIZ produced not only a prompt but also a later suppression of PTX-induced seizures. Although DIZ has effects on ATP-sensitive potassium (K(ATP)) channels when measured in vitro, our study suggests that additional mechanisms of action may involve the regulation of GABA transporters, which may aid in understanding epileptogenesis and inform investigators about future design and development of K(ATP) channel openers to treat epilepsy.