Effect of mutation of glycosylation sites on the Na+ dependence of steady-state and transient currents generated by the neuronal GABA transporter

Liu, Yanhong; Eckstein-Ludwig, Ursula; Fei, Jian; Schwarz, Wolfgang

doi:10.1016/s0005-2736(98)00200-4

Cited by 29 publications

(29 citation statements)

References 14 publications

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“…For the forward mode (i.e., the mode that describes removal of GABA from the extracellular space toward the cytoplasm and transporter re-orientation in the cell membrane), the initial estimates at 22°C suggested a value of 2.5 s −1 in Xenopus oocytes (Radian et al, 1986). This value agrees well with the turnover rate found by others in the same preparation and in similar experimental conditions: 5.8–7.6 s −1 at −60 mV (Eckstein-Ludwig et al, 1999), 6.3 s −1 at −60 mV (Liu et al, 1998), 6–13 s −1 at −80 mV (Mager et al, 1993), 13 s −1 at −40 mV (Bicho and Grewer, 2005). However, other reports have also estimated turnover rates at 37°C and −50–90 mV of 73–93 s −1 , much higher than it would be predicted by correcting the previous values for the estimated Q 10 value of 2.8 (Gonzales et al, 2007).…”

Section: The Turnover Rate Of Gaba Transporterssupporting

confidence: 92%

Structure, function, and plasticity of GABA transporters

Scimemi

2014

Front. Cell. Neurosci.

180

139

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GABA transporters belong to a large family of neurotransmitter:sodium symporters. They are widely expressed throughout the brain, with different levels of expression in different brain regions. GABA transporters are present in neurons and in astrocytes and their activity is crucial to regulate the extracellular concentration of GABA under basal conditions and during ongoing synaptic events. Numerous efforts have been devoted to determine the structural and functional properties of GABA transporters. There is also evidence that the expression of GABA transporters on the cell membrane and their lateral mobility can be modulated by different intracellular signaling cascades. The strength of individual synaptic contacts and the activity of entire neuronal networks may be finely tuned by altering the density, distribution and diffusion rate of GABA transporters within the cell membrane. These findings are intriguing because they suggest the existence of complex regulatory systems that control the plasticity of GABAergic transmission in the brain. Here we review the current knowledge on the structural and functional properties of GABA transporters and highlight the molecular mechanisms that alter the expression and mobility of GABA transporters at central synapses.

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Section: The Turnover Rate Of Gaba Transporterssupporting

confidence: 92%

Structure, function, and plasticity of GABA transporters

Scimemi

2014

Front. Cell. Neurosci.

180

139

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“…The time constant of the fluorescence change in NMDG-Cl buffer was around 80 ms and independent of membrane potential. This is compatible with the overall transport rate, estimated to be 5-10 s Ϫ1 (4,41). Nevertheless, this distinct C 6 7 C 0 transition rate might very well be highly dependent on the concentration of extracellular Na ϩ (as the apparent Na ϩ affinity of the first cation binding site is highly voltage dependent (42)) and therefore be much faster during GABA-transport.…”

Section: Discussionsupporting

confidence: 77%

Elucidating Conformational Changes in the γ-Aminobutyric Acid Transporter-1

Meinild

Loo

Skovstrup³

et al. 2009

Journal of Biological Chemistry

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The GABA transporter-1 (GAT-1) has three current-generating modes: GABA-coupled current, Li ؉ -induced leak current, and Na ؉ -dependent transient currents. We earlier hypothesized that Li ؉ is able to substitute for the first Na ؉ in the transport cycle and thereby induce a distinct conformation in GAT-1 and that the onset of the Li ؉ -induced leak current at membrane potentials more negative than ؊50 mV was due to a voltage-dependent conformational change of the Li ؉ -bound transporter. In this study, we set out to verify this hypothesis and seek insight into the structural dynamics underlying the leak current, as well as the sodium-dependent transient currents, by applying voltage clamp fluorometry to tetramethylrhodamine 6-maleimidelabeled GAT-1 expressed in Xenopus laevis oocytes. MTSET accessibility studies demonstrated the presence of two distinct conformations of GAT-1 in the presence of Na ␥-Aminobutyric acid (GABA) 2 is the major inhibitory neurotransmitter in the mammalian central nervous system. Continuous GABAergic neurotransmission is efficiently prevented by a GABA re-uptake system that transports GABA back into the synaptic processes via the GABA transporters (GAT). Four isoforms of the mammalian GAT have been found: GAT-1, GAT-2, GAT-3, and BGT-1 (betaine transporter-1) (1). These membrane proteins couple the transport of one GABA molecule to the transport of two Na ϩ and one Cl Ϫ (2, 3). Accordingly, the transport process is electrogenic and the transport activity can therefore be monitored by electrophysiological methods. GAT-1 has also been shown to generate: (i) an inwardly rectifying leak current in the presence of Li ϩ (and in complete absence of Na ϩ ) when the membrane potential is more negative than Ϫ50 mV (4 -6) and (ii) a presteady-state transient current in the presence of Na ϩ but in the absence of GABA in response to step jumps in membrane voltage (4, 7).GAT-1 is strictly dependent on external Na ϩ to drive the transport of GABA (7) and external GABA does not affect the Li ϩ -induced leak current (8). Taken together, this suggests that Li ϩ cannot induce the same conformation in GAT-1 as Na ϩ is capable of inducing: namely the conformation that is required for the binding and translocation of GABA. This is in contrast to, e.g. the related serotonin transporter and dopamine transporter in which substrate inhibits the Li ϩ -induced leak current (9, 10) and the Na ϩ /glucose cotransporter (SGLT1) where Li ϩ is able to sustain substrate transport (11). We have in an earlier study shown that Li ϩ is able to bind to the first low apparent affinity cation-binding site in the transport cycle (and replace Na ϩ ) but not to the second high apparent affinity cation-binding site (8). This finding was later confirmed by Kanner and co-workers (12), who were able to identify the cation-binding site with which Li ϩ interacts and is able to replace Na ϩ . According to our model, the transporter in the presence of Li ϩ is "stalled" in the conformation in which only the first cationbinding site is oc...

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“…In contrast, removal of glycosylation at N96 had no effect on plasma membrane expression, suggesting that deglycosylation at this position reduces the turnover number of the transporter. Similarly, deletion of glycosylation in the ␥-aminobutyric acid (GABA) transporter has been shown to reduce its turnover number (6,17). Removal of glycosylation at N71, N96, and N112 increased the affinity of OCT2 for TEA.…”

Section: Discussionmentioning

confidence: 99%

Functional influence ofN-glycosylation in OCT2-mediated tetraethylammonium transport

Pelis¹,

Suhre²,

Wright³

2006

American Journal of Physiology-Renal Physiology

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Pelis, Ryan M., Wendy M. Suhre, and Stephen H. Wright. Functional influence of N-glycosylation in OCT2-mediated tetraethylammonium transport. Am J Physiol Renal Physiol 290: F1118 -F1126, 2006. First published December 20, 2005 doi:10.1152/ajprenal.00462.2005.-OCT2, an organic cation transporter critical for removal of many drugs and toxins from the body, contains consensus sites for N-glycosylation at amino acid position 71, 96, and 112. However, the extent to which these sites are glycosylated by the cell, and the influence glycosylation has on OCT2 function, remains unknown. To address these issues, the acquisition of N-glycosylation was disrupted by mutating the amino acid asparagine (N) to glutamine (Q) at these sites in the rabbit ortholog of OCT2, which was expressed in Chinese hamster ovary cells. Disruption of N-glycosylation followed by Western blotting indicated that each site is indeed glycosylated and that OCT2 contains no other sites of Nglycosylation. Plasma membrane expression (determined by surface biotinylation) of the N112Q mutant, but not N71Q or N96Q mutants, was fourfold lower than that of wild-type OCT2, and unglycosylated OCT2 (N71Q/N96Q/N112Q) was sequestered in an unidentified intracellular compartment. The N71Q, N96Q, and N112Q mutants had a higher affinity (ϳ2-fold) for tetraethylammonium (TEA). Maximum transport rate was reduced in the N96Q (3-fold) and N112Q (5-fold) mutants, but not the N71Q mutant, and unglycosylated OCT2 failed to transport TEA (associated with its absence in the plasma membrane). Whereas the reduction in maximum transport rate of the N112Q mutant is consistent with its reduced plasma membrane expression, the lower rate of the N96Q mutant, which appeared to traffic properly, suggests that glycosylation at N96 increases the transporter turnover number. SLC22A2; organic cation; rabbit ORGANIC CATION TRANSPORTERS (OCTs) in the SLC22A gene family transport a variety of organic cations (OCs), including clinically important therapeutics (e.g., cimetidine) and environmental toxins (e.g., nicotine). Three OCTs (OCT1, OCT2, and OCT3) have been cloned, and each appears to be restricted to the basolateral membrane of barrier epithelia (32). In the renal proximal tubule, an important site for controlling plasma levels of OCs, all three OCTs are expressed and provide an essential pathway for OC uptake, the first step in tubular secretion (32). Depending on the prevailing set of electrical and chemical gradients, OCT-mediated transport can occur either via electrogenic facilitated diffusion (i.e., driven by the negative membrane potential) or OC/OC exchange (4, 5, 13). In humans, OCT2 (SLC22A2) has been postulated to be the greatest contributor to tubular OC secretion since its expression (as indicated by mRNA and protein levels) predominates over that of OCT1 and OCT3 (19). However, both OCT1 and OCT3 may play significant roles in the renal handling of select OCs (e.g., those transported inefficiently by OCT2) (32, 34). OCT1, 2, and 3 belong to a larger family of solute carr...

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Effect of mutation of glycosylation sites on the Na+ dependence of steady-state and transient currents generated by the neuronal GABA transporter

Cited by 29 publications

References 14 publications

Structure, function, and plasticity of GABA transporters

Structure, function, and plasticity of GABA transporters

Elucidating Conformational Changes in the γ-Aminobutyric Acid Transporter-1

Functional influence ofN-glycosylation in OCT2-mediated tetraethylammonium transport

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