LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen. (1/364)

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.  (+info)

Identification of the amine-polyamine-choline transporter superfamily 'consensus amphipathic region' as the target for inactivation of the Escherichia coli GABA transporter GabP by thiol modification reagents. Role of Cys-300 in restoring thiol sensitivity to Gabp lacking Cys. (2/364)

The Escherichia coli gamma-aminobutyric acid transporter GabP (gab permease) contains a functionally significant cysteine residue (Cys-300) within its consensus amphipathic region (CAR), a putative channel-forming structure that extends out of transmembrane helix 8 and into the adjoining cytoplasmic loop 8-9 of transporters from the amine-polyamine-choline (APC) superfamily. Here we show that of the five cysteine residues (positions 158, 251, 291, 300 and 443) in the E. coli GabP, Cys-300 is the one that renders the transport activity sensitive to inhibition by thiol modification reagents: whereas substituting Ala for Cys-300 mimics the inhibitory effect of thiol modification, substituting Ala at position 158, 251, 291 or 443 preserves robust transport activity and confers no resistance to thiol inactivation; and whereas the robustly active Cys-300 single-Cys mutant is fully sensitive to thiol modification, other single-Cys mutants (Cys at 158, 251, 291 or 443) exhibit kinetically compromised transport activities that resist further chemical inactivation by thiol reagents. The present study reveals additionally that Cys-300 exhibits (1) sensitivity to hydrophobic thiol reagents, (2) general resistance to bulky (fluorescein 5-maleimide) and/or charged {2-sulphonatoethyl methanethiosulphonate or [2-(trimethylammonium)ethyl] methanethiosulphonate} thiol reagents and (3) a peculiar sensitivity to p-chloromercuribenzenesulphonate (PCMBS). The accessibility of PCMBS to Cys-300 (located midway through the lipid bilayer) might be related to the structural similarity that it shares with guvacine (1, 2,3,6-tetrahydro-3-pyridinecarboxylic acid), a transported GabP substrate. These structural requirements for thiol sensitivity provide the first chemical evidence consistent with channel-like access to the polar surface of the CAR, a physical configuration that might provide a basis for understanding how this region impacts the function of APC transporters generally [Closs, Lyons, Kelly and Cunningham (1993) J. Biol. Chem. 268, 20796-20800] and the gab permease particularly [Hu and King (1998) Biochem. J. 300, 771-776].  (+info)

PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells. (3/364)

The PDZ target motifs located in the C-terminal end of many receptors and ion channels mediate protein-protein interactions by binding to specific PDZ-containing proteins. These interactions are involved in the localization of surface proteins on specialized membrane domains of neuronal and epithelial cells. However, the molecular mechanism responsible for this PDZ protein-dependent polarized localization is still unclear. This study first demonstrated that the epithelial gamma-aminobutyric acid (GABA) transporter (BGT-1) contains a PDZ target motif that mediates the interaction with the PDZ protein LIN-7 in Madin-Darby canine kidney (MDCK) cells, and then investigated the role of this interaction in the basolateral localization of the transporter. It was found that although the transporters from which the PDZ target motif was deleted were still targeted to the basolateral surface, they were not retained but internalized in an endosomal recycling compartment. Furthermore, an interfering BGT peptide determined the intracellular relocation of the native transporter. These data indicate that interactions with PDZ proteins determine the polarized surface localization of target proteins by means of retention and not targeting mechanisms. PDZ proteins may, therefore, act as a sort of membrane protein sorting machinery which, by recognizing retention signals (the PDZ target sequences), prevents protein internalization.  (+info)

Multiple G protein-coupled receptors initiate protein kinase C redistribution of GABA transporters in hippocampal neurons. (4/364)

Neurotransmitter transporters function in synaptic signaling in part through the sequestration and removal of neurotransmitter from the synaptic cleft. A recurring theme of transporters is that many can be functionally regulated by protein kinase C (PKC); some of this regulation occurs via a redistribution of the transporter protein between the plasma membrane and the cytoplasm. The endogenous triggers that lead to PKC-mediated transporter redistribution have not been elucidated. G-protein-coupled receptors that activate PKC are likely candidates to initiate transporter redistribution. We tested this hypothesis by examining the rat brain GABA transporter GAT1 endogenously expressed in hippocampal neurons. Specific agonists of G-protein-coupled acetylcholine, glutamate, and serotonin receptors downregulate GAT1 function. This functional inhibition is dose-dependent, mimicked by PKC activators, and prevented by specific receptor antagonists and PKC inhibitors. Surface biotinylation experiments show that the receptor-mediated functional inhibition correlates with a redistribution of GAT1 from the plasma membrane to intracellular locations. These data demonstrate (1) that endogenous GAT1 function can be regulated by PKC via subcellular redistribution, and (2) that signaling via several different G-protein-coupled receptors can mediate this effect. These results raise the possibility that some effects of G-protein-mediated alterations in synaptic signaling might occur through changes in the number of transporters expressed on the plasma membrane and subsequent effects on synaptic neurotransmitter levels.  (+info)

Passive water and ion transport by cotransporters. (5/364)

1. The rabbit Na+-glucose (SGLT1) and the human Na+-Cl--GABA (GAT1) cotransporters were expressed in Xenopus laevis oocytes, and passive Na+ and water transport were studied using electrical and optical techniques. Passive water permeabilities (Lp) of the cotransporters were determined from the changes in oocyte volume in response to osmotic gradients. The specific SGLT1 and GAT1 Lp values were obtained by measuring Lp in the presence and absence of blockers (phlorizin and SKF89976A). In the presence of the blockers, the Lp values of oocytes expressing SGLT1 and GAT1 were indistinguishable from the Lp of control oocytes. Passive Na+ transport (Na+ leak) was obtained from the blocker-sensitive Na+ currents in the absence of substrates (glucose and GABA). 2. Passive Na+ and water transport through SGLT1 were blocked by phlorizin with the same sensitivity (inhibitory constant (Ki), 3-5 microM). When Na+ was replaced with Li+, phlorizin also inhibited Li+ and water transport, but with a lower affinity (Ki, 100 microM). When Na+ was replaced by choline, which is not transported, the SGLT1 Lp was indistinguishable from that in Na+ or Li+, but in this case water transport was less sensitive to phlorizin. 3. The activation energies (Ea) for passive Na+ and water transport through SGLT1 were 21 and 5 kcal mol-1, respectively. The high Ea for Na+ transport is comparable to that of Na+-glucose cotransport and indicates that the process is dependent on conformational changes of the protein, while the low Ea for water transport is similar to that of water channels (aquaporins). 4. GAT1 also behaved as an SKF89976A-sensitive water channel. We did not observe passive Na+ transport through GAT1. 5. We conclude that passive water and Na+ transport through cotransporters depend on different mechanisms: Na+ transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel. In the case of SGLT1, we suggest that both the water channel and water cotransport could contribute to isotonic fluid transport across the intestinal brush border membrane.  (+info)

Coordinated transcriptional regulation of the unc-25 glutamic acid decarboxylase and the unc-47 GABA vesicular transporter by the Caenorhabditis elegans UNC-30 homeodomain protein. (6/364)

An important aspect of the specification of neuronal fate is the choice of neurotransmitter. In Caenorhabditis elegans the neurotransmitter GABA is synthesized by the UNC-25 glutamic acid decarboxylase (GAD) and packaged into synaptic vesicles by the UNC-47 transporter. Both unc-25 and unc-47 are expressed in 26 GABAergic neurons of five different types. Previously, we have identified that the unc-30 homeobox gene controls the fate of 19 type D GABAergic neurons. We report here that the UNC-30 homeodomain protein transcriptionally regulates the expression of unc-25 and unc-47 in the 19 type D neurons. UNC-30 bound to the unc-25 and unc-47 promoters sequence-specifically. Mutations in the UNC-30 binding sites of the unc-25 and unc-47 promoters abolished the expression of reporter genes in the D neurons. The ectopic expression of UNC-30 induced the ectopic expression of reporter genes driven by the wild-type unc-25 and unc-47 promoters. Our data establish a mechanism for cell type-specific transcriptional coregulation of genes required for the synthesis and packaging of the neurotransmitter GABA.  (+info)

The reactivity of the gamma-aminobutyric acid transporter GAT-1 toward sulfhydryl reagents is conformationally sensitive. Identification of a major target residue. (7/364)

The gamma-aminobutyric acid (GABA) transporter GAT-1 is a prototype of neurotransmitter transporters that maintain low synaptic levels of the transmitter. Transport by GAT-1 is sensitive to the polar sulfhydryl reagent 2-aminoethyl methanethiosulfonate. Following replacement of endogenous cysteines to other residues by site-directed mutagenesis, we have identified cysteine 399 as the major determinant of the sensitivity of the transporter to sulfhydryl modification. Cysteine-399 is located in the intracellular loop connecting putative transmembrane domains eight and nine. Binding of both sodium and chloride leads to a reduced sensitivity to sulfhydryl reagents, whereas subsequent binding of GABA increases it. Strikingly binding of the nontransportable GABA analogue SKF100330A gives rise to a marked protection against sulfhydryl modification. These effects were not observed in C399S transporters. Under standard conditions GAT-1 is almost insensitive toward the impermeant 2-(trimethylammonium)ethyl methanethiosulfonate. However, in a chloride-free medium, addition of SKF100330A renders wild type GAT-1, but not C399S, very sensitive to this impermeant reagent. These observations indicate that the accessibility of cysteine 399 is highly dependent on the conformation of GAT-1. Consequently, topological assignments based on accessibility of endogeneous or engineered cysteines to small polar sulfhydryl reagents need to be interpreted with extreme caution.  (+info)

GAT1 (GABA:Na+:Cl-) cotransport function. Steady state studies in giant Xenopus oocyte membrane patches. (8/364)

Neurotransmitter transporters are reported to mediate transmembrane ion movements that are poorly coupled to neurotransmitter transport and to exhibit complex "channel-like" behaviors that challenge the classical "alternating access" transport model. To test alternative models, and to develop an improved model for the Na+- and Cl--dependent gamma-aminobutyric acid (GABA) transporter, GAT1, we expressed GAT1 in Xenopus oocytes and analyzed its function in detail in giant membrane patches. We detected no Na+- or Cl--dependent currents in the absence of GABA, nor did we detect activating effects of substrates added to the trans side. Outward GAT1 current ("reverse" transport mode) requires the presence of all three substrates on the cytoplasmic side. Inward GAT1 current ("forward" transport mode) can be partially activated by GABA and Na+ on the extracellular (pipette) side in the nominal absence of Cl-. With all three substrates on both membrane sides, reversal potentials defined with specific GAT1 inhibitors are consistent with the proposed stoichiometry of 1GABA:2Na+:1Cl-. As predicted for the "alternating access" model, addition of a substrate to the trans side (120 mM extracellular Na+) decreases the half-maximal concentration for activation of current by a substrate on the cis side (cytoplasmic GABA). In the presence of extracellular Na+, the half-maximal cytoplasmic GABA concentration is increased by decreasing cytoplasmic Cl-. In the absence of extracellular Na+, half-maximal cytoplasmic substrate concentrations (8 mM Cl-, 2 mM GABA, 60 mM Na+) do not change when cosubstrate concentrations are reduced, with the exception that reducing cytoplasmic Cl- increases the half-maximal cytoplasmic Na+ concentration. The forward GAT1 current (i.e., inward current with all extracellular substrates present) is inhibited monotonically by cytoplasmic Cl- (Ki, 8 mM); cytoplasmic Na+ and cytoplasmic GABA are without effect in the absence of cytoplasmic Cl-. In the absence of extracellular Na+, current-voltage relations for reverse transport current (i.e., outward current with all cytoplasmic substrates present) can be approximated by shallow exponential functions whose slopes are consistent with rate-limiting steps moving 0.15-0.3 equivalent charges. The slopes of current-voltage relations change only little when current is reduced four- to eightfold by lowering each cosubstrate concentration; they increase twofold upon addition of 100 mM Na+ to the extracellular (pipette) side.  (+info)