Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2.

The neurotransmitter glycine is removed from the synaptic cleft by two Na(+)-and Cl(-)-dependent transporters, the glial (GLYT1) and neuronal (GLYT2) glycine transporters. GLYT2 lacks a conserved cysteine in the first hydrophilic loop (EL1) that is reactive to [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) in related transporters. A chimeric GLYT2 (GLYT2a-EL1) that contains GLYT1 sequences in this region, including the relevant cysteine, was sensitive to the reagent, and its sensitivity was decreased by co-substrates. We combined cysteine-specific biotinylation to detect transporter-reagent interactions with MTSET inactivation assays and temperature dependence analysis to study the mechanism by which Cl(-), Na(+), and glycine reduce methanethiosulfonate reagent inhibition. We demonstrate a Na(+) protective effect rather than an increased susceptibility to the reagent exerted by Li(+), as reported for the serotonin transporter. The different inhibition, protection, and reactivation properties between GLYT2a-EL1 and serotonin transporter suggest that EL1 is a source of structural heterogeneity involved in the specific effect of lithium on serotonin transport. The protection by Na(+) or Cl(-) on GLYT2a-EL1 was clearly dependent on temperature, suggesting that EL1 is not involved in ion binding but is subjected to ion-induced conformational changes. Na(+) and Cl(-) were required for glycine protection, indicating the necessity of prior ion interaction with the transporter for the binding of glycine. We conclude that EL1 acts as a fluctuating hinge undergoing sequential conformational changes during the transport cycle.

The glial and the neuronal glycine transporters differ in their reactivity to sulfhydryl reagents.

The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na(+)/Cl(-)-dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na(+) coupling. We have characterized under voltage clamp their reactivity toward the membrane impermeant sulfhydryl reagent [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). In Xenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na(+)-dependent charge movement, the glycine-evoked current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and GlyT2a-EL1) resulting in AFQ <--> CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na(+), Cl(-), and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation.

The role of N-glycosylation in transport to the plasma membrane and sorting of the neuronal glycine transporter GLYT2.

Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35-40% after enzymatic deglycosylation of the transporter reconstituted into liposomes. Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellular loop of GLYT2, produced an inactive protein that was retained in intracellular compartments when transiently transfected in COS cells or in nonpolarized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 localizes in the apical surface as assessed by transport and biotinylation assays. However, a partially unglycosylated mutant (triple mutant) was distributed in a nonpolarized manner in MDCK cells. The apical localization of GLYT2 occurred by a glycolipid rafts independent pathway.

Differential effects of ethanol on glycine uptake mediated by the recombinant GLYT1 and GLYT2 glycine transporters.

The effects of ethanol on the function of recombinant glycine transporter 1 (GLYT1) and glycine transporter 2 (GLYT2) have been investigated. GLYT1b and GLYT2a isoforms stably expressed in human embryonic kidney 293 (HEK 293) cells showed a differential behaviour in the presence of ethanol; only the GLYT2a isoform was acutely inhibited. The 'cut-off' (alcohols with four carbons) displayed by the n-alkanols on GLYT2a indicates that a specific binding site for ethanol exists on GLYT2a or on a GLYT2a-interacting protein. The non-competitive inhibition of GLYT2a indicates an allosteric modulation by ethanol of GLYT2a activity. Chronic treatment with ethanol caused differential adaptive responses on the activity and the membrane expression levels of these transporters. The neuronal GLYT2a isoform decreased in activity and surface expression and the mainly glial GLYT1b isoform slightly increased in function and surface density. These changes may be involved in some of the modifications of glycinergic or glutamatergic neurotransmitter systems produced by ethanol intoxication.

Polarized distribution of glycine transporter isoforms in epithelial and neuronal cells.

Asymmetrical distribution of Na(+)- and Cl(-)-dependent neurotransmitter transporters on the cell surface of polarized cells seems to be a generalized feature in this gene family. In the present study we analyzed the subcellular distribution of the various isoforms of the glycine transporters GLYT1 and GLYT2 after heterologous expression in polarized MDCK cells and in hippocampal neurons. Our results indicate that glycine transporters are asymmetrically distributed in an isoform- and cell-type-specific manner. GLYT1b is localized in the basolateral and somatodendritic domains of MDCK cells and neurons, respectively. However, GLYT1a is somatodendritic in neurons but is predominantly expressed in the apical surface of MDCK cells. The two isoforms of GLYT2 (GLYT2a and GLYT2b) are found at the apical surface in epithelial cells but are uniformly distributed in neurons. By using site-directed mutagenesis we have been able to identify signals for basolateral/somatodendritic localization in the amino-terminal region of GLYT1 and in two dileucine motifs located in the carboxyl tail of this protein. These results contribute to defining the mechanisms of asymmetrical distribution of transporters on the cell surface of polarized cells.

Differential properties of two stably expressed brain-specific glycine transporters.

Clonal cell lines stably expressing the glial glycine transporter 1b (GLYT1b) and the neuronal glycine transporter 2 (GLYT2) from rat brain have been generated and used comparatively to examine their kinetics, ion dependence, and electrical properties. Differential sensitivity of the transporters to sarcosine is clearly exhibited by the clonal cell lines. GLYT2 transports glycine with higher apparent affinity than GLYT1b and is not inhibited by any assayed compound, as deduced by glycine transport assays and electrophysiological recordings. A sigmoidal Na+ dependence of the glycine uptake by the stable cell lines is observed, indicating the involvement of more than one Na+ in the transport process. A more cooperative behavior for Na+ of GLYT2 than GLYT1b is suggested. One Cl- is required for GLYT1b and GLYT2 transport cycles, although GLYT1b shows three times higher affinity for this ion than GLYT2. The number of expressed transporters was sufficient to allow electrophysiological recordings of the uptake current in the two stable cell lines. GLYT2 exhibits more voltage dependence in both its glycine-evoked current and its capacitive currents recorded in the absence of substrate.