[Show abstract][Hide abstract] ABSTRACT: The functional properties of an ortholog of the B(0)AT1 (SLC6A19) amino acid transporter, cloned from the intestine of the sea bass Dicentrachus labrax, were investigated. The two-electrode voltage-clamp technique was applied to Xenopus laevis oocytes heterologously expressing the transporter in order to measure the currents associated with the transport process in different conditions. In particular the substrate specificity, the ionic requirements, and possible effects of pH were examined. Among the organic substrates, leucine, glycine, serine and valine generated the largest transport currents with apparent affinities in the lower millimolar range. The importance of Na(+) as the driver ion in the transport process is confirmed, although Li(+) is also capable to sustain transport, while K(+) is not. No evidence of a relevant role of Cl(-) in the transport activity was found. Concerning the other two kinds of currents commonly found in electrogenic transporters, very fast presteady-state currents were detected in the absence of organic substrate, while lithium-specific leak currents were not observed. The comparison of these properties with those of the mammalian and insect orthologs may give interesting indication for future structure-function studies in this transporter subfamily.
Full-text · Article · Jul 2013 · Comparative biochemistry and physiology. Part A, Molecular & integrative physiology
[Show abstract][Hide abstract] ABSTRACT: During digestion, dietary proteins cleaved in di and tri- peptides are translocated from the intestinal lumen into the enterocytes via PepT1 (SLC15A1) using an inwardly directed proton electrochemical gradient. The kinetic properties in various PepT1 orthologs (Dicentrarchus labrax, Oryctolagus cuniculus, Danio rerio) have been explored to determine the transport efficiency of different combinations of lysine, methionine, and glycine. Species-specific differences were observed. Lys-Met resulted the best substrate at all tested potentials in sea bass and rabbit PepT1, whereas in the zebrafish transporter all tested dipeptides (except Gly-Lys) elicited similar currents independently on the charge position or amino acid composition. For the sea bass and rabbit PepT1, kinetic parameters, K(05) and I(max) and their ratio, show the importance of the position of the charged lysine in the peptide. The PepT1transporter of these species have very low affinity for Lys-Lys and Gly-Lys; this reduces the transport efficiency which is instead higher for Lys-Met and Lys-Gly. PepT1 from zebrafish showed relatively high affinity and excellent transport efficiency for Met-Lys and Lys-Met. These data let us to speculate about the structural determinants involved in substrate interaction according to the model proposed for this transporter.
Full-text · Article · Dec 2012 · Comparative biochemistry and physiology. Part A, Molecular & integrative physiology
[Show abstract][Hide abstract] ABSTRACT: The effects of temperature on the operation of two ion-coupled cotransporters of the SLC6A family, namely rat GAT1 (SLC6A1) and KAAT1 (SLC6A19) from Manduca sexta, have been studied by electrophysiological means in Xenopus laevis oocytes expressing these proteins. The maximal transport-associated current (I(max)) and the apparent substrate affinity (K(05)) were measured. In addition to the expected increase in transport rate (Q(10) = 3-6), both transporters showed greater K(05) values (i.e., a decrease in apparent affinity) at higher temperatures. The transport efficiency, estimated as I(max)/K(05), increased at negative potentials in both transporters, but did not show statistically significant differences with temperature. The observation that the apparent substrate affinity is inversely related to the transport rate suggests a kinetic regulation of this parameter. Furthermore, the present results indicate that the affinities estimated at room temperature for mammalian cotransporters may not be simply extrapolated to their physiological operating conditions.
Full-text · Article · Dec 2012 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: The effects of temperature on the functional properties of the intestinal oligopeptide transporter PepT1 from rabbit have been investigated using electrophysiological methods. The dipeptide Gly–Gln at pH 6.5 or 7.5 was used as substrate. Raising the temperature in the range 20–30 °C causes an increase in the maximal transport-associated current (I
max) with a Q
10 close to 4. Higher temperatures accelerate the rate of decline of the presteady-state currents observed in the absence of organic substrate. The voltage dependencies of the intramembrane charge movement and of the time constant of decline are both shifted towards more negative potentials by higher temperatures. The shift is due to a stronger action of temperature on the outward rate of charge movement compared to the inward rate, indicating a lower activation energy for the latter process. Consistently, the activation energy for the complete cycle is similar to that of the inward rate of charge movement. Temperature also affects the binding rate of the substrate: the K
0.5–V curve is shifted to more negative potentials by higher temperatures, resulting in a lower apparent affinity in the physiological range of potentials. The overall efficiency of transport, estimated as the I
0.5 ratio is significantly increased at body temperature.
No preview · Article · Jun 2012 · Pflügers Archiv - European Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: The role of internal substrates in the biophysical properties of the GABA transporter GAT1 has been investigated electrophysiologically in Xenopus oocytes heterologously expressing the cotransporter. Increments in Cl(-) and/or Na(+) concentrations caused by intracellular injections did not produce significant effects on the pre-steady-state currents, while a positive shift of the charge-voltage (Q-V) and decay time constant (τ)-voltage (τ-V) curves, together with a slowing of τ at positive potentials, was observed following treatments producing cytosolic Cl(-) depletion. Activation of the reverse transport mode by injections of GABA caused a reduction in the displaced charge. In the absence of external Cl(-), a stronger reduction in the displaced charge, together with a significant increase in reverse transport current, was observed. Therefore, complementarity between pre-steady-state and transport currents, observed in the forward mode, is preserved in the reverse mode. All these findings can be qualitatively reproduced by a kinetic scheme in which, in the forward mode, the Cl(-) ion is released first, after the inward charge movement, while the two Na(+) ions can be released only after binding of external GABA. In the reverse mode, internal GABA must bind first to the empty transporter, followed by internal Na(+) and Cl(-).
No preview · Article · Dec 2011 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: The role of intracellular ions on the reverse GABA transport by the neuronal transporter GAT1 was studied using voltage-clamp and [(3)H]GABA efflux determinations in Xenopus oocytes transfected with heterologous mRNA. Reverse transport was induced by intracellular GABA injections and measured in terms of the net outward current generated by the transporter. Changes in various intracellular ionic conditions affected the reverse current: higher concentrations of Na(+) enhanced the ratio of outward over inward transport current, while a considerable decrease of the outward current and a parallel reduction of the transporter-mediated GABA efflux were observed after treatments causing a diminution of the intracellular Cl(-) concentration. Particularly interesting was the impairment of the reverse transport observed after depletion of internal Cl(-) generated by the activity of a coexpressed K(+)-Cl(-) exporter KCC2. This finding suggests that reverse GABA transport may be physiologically regulated during early neuronal development, similarly to the functional alterations seen in GABA receptors caused by KCC2 activity.
[Show abstract][Hide abstract] ABSTRACT: Background / Purpose:
Neurotransmitters are normally taken up in neurons and glial cells through the transporter proteins using a sodium and chloride electrochemical gradient. These neurotransmitter transporters (NSS) are essential for the correct cellular communication. A reverse transport, i.e. the efflux of neurotransmitter via the same protein, have been previously reported, and a role for this process in various neurological disorders has been hypothesized.Reverse GABA transport was induced in Xenopus oocytes heterologously expressing the rat neuronal transporter rGAT1, by intracellular GABA injections. By measurement of the outward transport currents in a voltage-clamp and by [3H] GABA efflux determinations, reverse transport was estimated. Differences in the reverse currents were highlighted by changing intracellular ionic conditions. Higher concentrations of Na enhanced the ratio of outward to inward transport current. Treatments causing a reduction of the intracellular Cl concentration decreased the outward current. The depletion of internal Cl was achieved by the coexpression of ion cotransporter KCC2 (KCl cotransporter) and induced a significant decrease in the relative reverse transport current.
This finding suggests that reverse GABA transport may be physiologically regulated by KCC2 activity during early neuronal development or in injury and epilepsy, similar to GABA receptors. In addition, any other conditions favouring higher intracellular chloride levels, such as under-expression of chloride exporters, defects in osmoregulation or in ionic homeostasis, may be considered potentially relevant in affecting the balance between forward and reverse mode of neurotransmitter transport.
[Show abstract][Hide abstract] ABSTRACT: The oligopeptide transporter PepT1 is a protein found in the membrane of the cells of the intestinal walls, and represents the main route through which proteic nutrients are absorbed by the organism. Along the polypeptidic chain of this protein, two oppositely charged amino acids, an arginine in position 282 and an aspartate in position 341 of the sequence, have been hypothesised to form a barrier in the absorption pathway. In this paper we show that appropriate mutations of these amino acids change the properties of PepT1 in a way that confirms that these parts of the protein indeed act as an electrostatic gate in the transport process. The identification of the structural basis of the functional mechanism of this transporter is important because, in addition to its role in nutrient uptake, PepT1 represents a major pathway for the absorption of several therapeutic drugs.
Abstract The effects of mutations in the charge pair residues Arg282 and Asp341 of the rabbit oligopeptide transporter PepT1 have been studied using electrophysiology in mRNA-injected Xenopus oocytes. Substitution of Arg282 with neutral or negatively charged residues produced a shift towards more positive potentials in the characteristics of charge movement with respect to the wild-type form. Conversely replacement of Asp341 with Arg reduced both pre-steady-state and transport currents and produced a negative shift of the charge movement properties. Both kinds of currents remained pH-sensitive in the mutants. All functional mutants were correctly localized on the cell membrane. Removal of the positive charge of Arg282 produced transporters able to generate conspicuous outward currents whose reversal potential was affected by external pH and by substrate concentration. This suggests that the mutants still translocate protons and substrate as a complex. Charged substrates were accepted by the mutants with the same potency order as the wild-type. The results support the idea that Arg282 and Asp341 play the role of electrostatic gates in the PepT1 transport cycle.
Full-text · Article · Feb 2011 · The Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: Electrophysiological and biophysical analyses were used to compare the partial and complete transport cycles of the intestinal oligopeptide transporter PepT1 among three species (seabass, zebrafish and rabbit). On the whole, the presteady-state currents of the fish transporters were similar to each other. Rabbit PepT1 differed from the fish transporters by having slower-decaying currents, and the charge vs. potential (Q/V) and time constant vs. potential (τ/V) curves shifted to more positive potentials. All of the isoforms were similarly affected by external pH, showing acidity-induced slowing of the transients and positive shifts in the Q/V and τ/V curves. Analysis of the pH-dependence of the unidirectional rates of the intramembrane charge movement suggested that external protonation of the protein limits the speed of this process in both directions. The complete cycle of the transporter was studied using the neutral dipeptide Gly-Gln. Michaelis-Menten analysis confirmed that, in all species, acidity significantly increases the apparent affinity for the substrate but does not strongly impact maximal transport current. Simulations using a kinetic model incorporating the new findings showed good agreement with experimental data for all three species, both with respect to the presteady-state and the transport currents.
Full-text · Article · Jan 2011 · Channels (Austin, Tex.)
[Show abstract][Hide abstract] ABSTRACT: The functional and structural basis of reverse operation of PepT1 has been studied in Xenopus oocytes expressing the wild-type and mutated forms of this protein. Using brief pulses from a negative holding potential, wild-type and Arg282 mutants exhibit outward currents in the presence of Gly-Gln. The reversal potential of these currents is affected by both pH and substrate concentration, confirming coupled transport in the wild type and in the mutants as well. Long-lasting voltage and current-clamp experiments show that the outward currents are only temporary, and reflect accumulation and/or depletion effects near the membrane. The ability to operate in reverse mode was confirmed in all isoforms by intracellular injection of substrate. The role of Arg282 and Asp341 in the reverse transport was also investigated using charged substrates. Positive Lys-Gly (but not Gly-Lys) showed enhanced transport currents in the Arg282 mutants. In contrast, negative Gly-Asp and Asp-Gly elicited modest currents in all isoforms.
No preview · Article · Dec 2010 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: The relation between presteady-state (transient) currents elicited by voltage steps in the absence of organic substrate and transport-associated currents in the presence of glycine was investigated in Xenopus oocytes expressing the neuronal glycine transporter GlyT1b. Saturating amounts of glycine converted the transient currents in steady transport currents. Analysis of the transient currents abolished by the substrate confirmed the intramembrane nature of the underlying charge movement process. The sigmoidal Q/V relationship had a moderate slope consistent with the known GlyT1b stoichiometry. The transient currents were best fitted by the sum of two exponentials, with the slow time constant (τ
slow) being in the order of tens of milliseconds. The apparent affinity for glycine was in the micromolar range and voltage-dependent, slightly decreasing at positive potentials. Numerical simulations show that a simplified, three-state model is sufficient to explain the main features of GlyT1b operation.
No preview · Article · Sep 2009 · Journal of Molecular Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Complementary RNA, derived from the intestine of the sea bass Dicentrarchus labrax and putatively coding for a pH-dependent oligopeptide transporter PepT1 (SLC15 family), was injected in Xenopus oocytes that were subsequently tested with electrophysiological techniques. Transport-associated currents were observed when various di- or tripeptides were applied at concentrations ranging between 0.1 and 10 mM. No currents were generated by histidine nor by other single amino acids. Sea bass PepT1 also exhibited presteady-state currents in the absence of substrates. Acidic pH slowed down the relaxation time constant of these currents and shifted both Q/V and tau/V relationships toward more positive voltages. Michaelis-Menten analysis of the transport currents showed an increase in apparent substrate affinity at acidic pH, which was very similar to that exhibited by the related transporter from zebrafish (Danio rerio), but in contrast, did not demonstrate a significant effect of pH on the maximal transport current.
No preview · Article · Aug 2009 · Pflügers Archiv - European Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: KAAT1 and CAATCH1 are amino acid transporters cloned from the intestine of the lepidoptera Manduca sexta. They are members of the SLC6/NSS family, which groups membrane proteins that use Na(+), K(+), and Cl⁻ gradients for the coupled transport of amines and amino acids. The report of the atomic-resolution x-ray crystal structure of the eubacterium Aquifex aeolicus leucine transporter (AaLeuT) has contributed significantly to understanding of the structure-function relationship in NSS proteins. Transport by AaLeuT is Cl⁻ independent, whereas many neurotransmitter:sodium symporters like serotonin transporter (SERT), GABA transporter (GAT1), dopamine transporter, and norephinephrine transporter, among others, are strongly Cl⁻ dependent.
Full-text · Article · Oct 2008 · Channels (Austin, Tex.)
[Show abstract][Hide abstract] ABSTRACT: KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K(+) and Li(+) in addition to Na(+). We have previously demonstrated that Asp338 is essential for KAAT1 activation by K(+) and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis, and expression in Xenopus laevis oocytes, we identified Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both Na(+) and K(+). However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na(+)-dependent transport and the block in K(+)-dependent transport that characterize the D338E mutant. K(+)-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)(3), we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus both structural and functional evidence suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na(+) binding site in the recently solved crystal structure of the NSS transporter LeuT(Aa) (41). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway.
Full-text · Article · Nov 2007 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: The substrate specificity of KAAT1, a Na+- and K+-dependent neutral amino acid cotransporter cloned from the larva of the invertebrate Manduca sexta and belonging to the SLC6A gene family has been investigated using electrophysiological and radiotracer methods. The specificity of KAAT1 was compared to that of CAATCH1, a strictly related transporter with different amino acid selectivity. Competition experiments between different substrates indicate that both transporters bind leucine more strongly than threonine and proline, the difference between KAAT1 and CAATCH1 residing in the incapacity of the latter to complete the transport cycle in presence of leucine. The behaviour of CAATCH1 is mimicked by the S308T mutant form of KAAT1, constructed on the basis of the atomic structure of a leucine-transporting bacterial member of the family, which indicates the participation of this residue in the leucine-binding site. The reverse mutation T308S in CAATCH1 conferred to this transporter the ability to transport leucine in presence of K+. These results may be interpreted by a kinetic scheme in which, in presence of Na+, the leucine-bound state of the transporter is relatively stable, while in presence of K+ and at negative potentials the progression of the leucine-bound form along the cycle is favoured. In this context serine 308 appears to be important in allowing the change to the inward-facing conformation of the transporter following substrate binding, rather than in determining the binding specificity.
Full-text · Article · Jul 2007 · The Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: The highly homologous neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the Manduca sexta larva, are members of the Na(+)/Cl(-)-dependent transporter family. Recent evidence indicates that transporters of this family form constitutive oligomers. CAATCH1 and KAAT1 give rise to specific kinds of current depending on the transported amino acid, cotransported ion, pH, and membrane voltage. Different substrates induce notably distinct transport-associated currents in the two proteins that represent useful tools in structural-functional studies. To determine whether KAAT1 and CAATCH1 form functional oligomers, we have constructed four concatameric proteins for electrophysiological analysis, consisting of one KAAT1 protein covalently linked to another KAAT1 (K-K concatamer) or to CAATCH1 (K-C concatamer) and vice versa (C-C concatamer and C-K concatamer), and eight constructs where the two transporters were linked to yellow or cyan fluorescent protein in the NH(2) or COOH terminus, to determine the oligomer formation and the relative distance between the different subunits by fluorescence resonance energy transfer (FRET) analysis. Heterologous expression of the concatenated constructs and coinjection of the original proteins in different proportions allowed us to compare the characteristics of the currents to those of the oocytes expressing only the wild-type proteins. All the constructs were fully active, and their electrophysiological behavior was consistent with the activity as monomeric proteins. However, the FRET studies indicate that these transporters form oligomers in agreement with the LeuT(Aa) atomic structure and confirm that the COOH termini of the adjacent subunits are closer than NH(2) termini.
Preview · Article · May 2007 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: Up to now, baclofen (a GABA(B) receptor agonist) has been used for the treatment of severe spasticity unresponsive to oral antispasmodics. Although in humans it is usually administered at 2 mg/ml, the dosage to be used in the treatment of other diseases is unknown. For this reason, it is important to determine the safe maximum dosage and toxicity at the clinically used concentration. Primary cortical neurons represent a useful model to test the safety of baclofen. We performed a colorimetric assay (MTT test) as well as electron microscopy investigations, to determine neuronal survival after the treatment with baclofen at a concentration of 2 and 4 mg/ml. Our results demonstrated that, in our experimental model, neither concentration affected neuronal survival. Considering the above results, we can conclude that at the used concentrations, this drug is safe and its clinical use should be encouraged.
No preview · Article · Dec 2006 · European Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: We investigated the role of the Q291 glutamine residue in the functioning of the rat gamma-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter.
Full-text · Article · Feb 2006 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently.
No preview · Article · Jan 2006 · Cellular and Molecular Life Sciences CMLS
[Show abstract][Hide abstract] ABSTRACT: 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 (alpha+beta), while the Na(+) apparent affinity is directly related to their ratio (beta/alpha). 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.
Full-text · Article · Jan 2005 · The Journal of Physiology