Glutamate modulation of GABA transport in retinal horizontal cells of the skate.
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Transport of the amino acid GABA into neurons and glia plays a key role in regulating the effects of GABA in the vertebrate retina. We have examined the modulation of GABA-elicited transport currents of retinal horizontal cells by glutamate, the likely neurotransmitter of vertebrate photoreceptors. Enzymatically isolated external horizontal cells of skate were examined using whole-cell voltage-clamp techniques. GABA (1 mM ) elicited an inward current that was completely suppressed by the GABA transport inhibitors tiagabine (10 microM) and SKF89976-A (100 microM), but was unaffected by 100 microM picrotoxin. Prior application of 100 microM glutamate significantly reduced the GABA-elicited current. Glutamate depressed the GABA dose-response curve without shifting the curve laterally or altering the voltage dependence of the current. The ionotropic glutamate receptor agonists kainate and AMPA also reduced the GABA-elicited current, and the effects of glutamate and kainate were abolished by the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline. NMDA neither elicited a current nor modified the GABA-induced current, and metabotropic glutamate analogues were also without effect. Inhibition of the GABA-elicited current by glutamate and kainate was reduced when extracellular calcium was removed and when recording pipettes contained high concentrations of the calcium chelator BAPTA. Caffeine (5 mM) and thapsigargin (2 nM), agents known to alter intracellular calcium levels, also reduced the GABA-elicited current, but increases in calcium induced by depolarization alone did not. Our data suggest that glutamate regulates GABA transport in retinal horizontal cells through a calcium-dependent process, and imply a close physical relationship between calcium-permeable glutamate receptors and GABA transporters in these cells. (+info)
The anticonvulsant valproate increases the turnover rate of gamma-aminobutyric acid transporters.
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Valproate is an important anticonvulsant currently in clinical use for the treatment of seizures. We used electrophysiological and tracer uptake methods to examine the effect of valproate on a gamma-aminobutyric acid (GABA) transporter (mouse GAT3) expressed in Xenopus laevis oocytes. In the absence of GABA, valproate (up to 50 mm) had no noticeable effect on the steady-state electrogenic properties of mGAT3. In the presence of GABA, however, valproate enhanced the GABA-evoked steady-state inward current in a dose-dependent manner with a half-maximal concentration of 4.6 +/- 0.5 mm. Maximal enhancement of the GABA-evoked current was 275 +/- 10%. Qualitatively similar observations were obtained for human GAT1 and mouse GAT4. The valproate enhancement did not alter the Na(+) or Cl(-) dependence of the steady-state GABA-evoked currents. Uptake experiments under voltage clamp suggested that the valproate enhancement of the GABA-evoked current was matched by an enhancement in GABA uptake. Thus, despite the increase in GABA-evoked current, ion/GABA co-transport remained tightly coupled. Uptake experiments indicated that valproate is not transported by mouse GAT3 in the absence or presence of GABA. Valproate also enhanced the rate of the partial steps involved in transporter presteady-state charge movements. We propose that valproate increases the turnover rate of GABA transporters by an allosteric mechanism. The data suggest that at its therapeutic concentration, valproate may enhance the activity of neuronal and glial GABA transporters by up to 10%. (+info)
Vigabatrin induces tonic inhibition via GABA transporter reversal without increasing vesicular GABA release.
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Two forms of GABAergic inhibition coexist: fast synaptic neurotransmission and tonic activation of GABA receptors due to ambient GABA. The mechanisms regulating ambient GABA have not been well defined. Here we examined the role of the GABA transporter in the increase in ambient [GABA] induced by the anticonvulsant vigabatrin. Pretreatment of cultured rat hippocampal neurons with vigabatrin (100 microM) for 2-5 days led to a large increase in ambient [GABA] that was measured as the change in holding current induced by bicuculline during patch-clamp recordings. In contrast, there was a decrease in the frequency of spontaneous miniature inhibitory postsynaptic currents mIPSCs with no change in their amplitude distribution, and a decrease in the magnitude of IPSCs evoked by presynaptic stimulation during paired recordings. The increase in ambient [GABA] was not prevented by blockade of vesicular GABA release with tetanus toxin or removal of extracellular calcium. During perforated patch recordings, the increase in ambient [GABA] was prevented by blocking the GABA transporter, indicating that the GABA transporter was continuously operating in reverse and releasing GABA. In contrast, blocking the GABA transporter increased ambient [GABA] during whole cell patch-clamp recordings unless GABA and Na(+) were added to the recording electrode solution, indicating that whole cell recordings can lead to erroneous conclusions about the role of the GABA transporter in control of ambient GABA. We conclude that the equilibrium for the GABA transporter is a major determinant of ambient [GABA] and tonic GABAergic inhibition. We propose that fast GABAergic neurotransmission and tonic inhibition can be independently modified and play complementary roles in control of neuronal excitability. (+info)
Plasma membrane GABA transporters reside on distinct vesicles and undergo rapid regulated recycling.
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Plasma membrane neurotransmitter transporters affect synaptic signaling through transmitter sequestration. Transporters redistribute to and from the plasma membrane, suggesting a role for trafficking in regulating synaptic transmitter levels. One method for controlling transmitter levels would be to regulate transporter redistribution in parallel with transmitter release. Thus, how similar are these processes? We show that the trafficking of the GABA transporter GAT1 resembles the trafficking of neurotransmitter-filled synaptic vesicles: (1) transporters located on the plasma membrane are internalized and reinserted into the plasma membrane on the order of minutes; (2) the rate of recycling is depolarization and calcium dependent; (3) GAT1 internalization is associated with clathrin and dynamin; and (4) intracellular GAT1 is associated with multiple compartments and, more importantly, is found on a distinct class of vesicles. These vesicles are clear, approximately 50 nm in diameter, and contain many proteins found on neurotransmitter-containing small synaptic vesicles; however, they appear to lack several traditional small synaptic vesicle proteins, such as synaptophysin and the vesicular GABA transporter. These data provide additional support for the hypothesis that GABA transporters traffic in parallel with neurotransmitter-containing small synaptic vesicles and also raise the possibility that some fraction of vesicles found in GABAergic neurons may not be participating in transmitter release but rather in the rapid regulated redistribution of membrane proteins involved in transmitter uptake. (+info)
Synapse density regulates independence at unitary inhibitory synapses.
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Neurotransmitter transporters may promote synapse specificity by limiting spillover between release sites. At GABAergic synapses, transport block prolongs synaptic responses when many inputs are activated, yet it is unclear whether transporters alter signaling by single axons. We found that unitary IPSCs generated by paired recordings between hippocampal interneurons and granule cells could be either prolonged or totally unaffected by block of GABA transporters. This variability was explained by the density of active release sites rather than the number of active sites. Prolongation by transport block required release from multiple sites and was enhanced by repetitive activation. Furthermore, transport-sensitive unitary IPSCs were accelerated when the release probability was reduced, indicating that cross talk prolonged the time course of IPSCs even when transport was intact. Our results suggest that the release site density regulates the degree of cross talk as well as the contribution of transporters to GABA clearance. Thus, interplay between release site density and transporter action determines the independence of unitary inhibitory synapses. (+info)
Spontaneous seizures and loss of axo-axonic and axo-somatic inhibition induced by repeated brief seizures in kindled rats.
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Repeated brief seizures evoked by kindling progressively increase seizure susceptibility and eventually induce spontaneous seizures. Previous studies have demonstrated that the initial seizures evoked by kindling increase paired-pulse inhibition at 15-25 msec interpulse intervals in the dentate gyrus and also induce apoptosis, progressive neuronal loss, mossy fiber sprouting, and neurogenesis, which could potentially alter the balance of excitation and/or inhibition and modify functional properties of hippocampal circuits. In these experiments, paired-pulse inhibition in the dentate gyrus was reduced or lost after approximately 90-100 evoked seizures in association with emergence of spontaneous seizures. Evoked IPSCs examined by single electrode voltage-clamp methods in granule cells from kindled rats experiencing spontaneous seizures demonstrated altered kinetics (reductions of approximately 48% in 10-90% decay time, approximately 40% in tau, and approximately 65% in charge transfer) and confirmed that decreased inhibition contributed to the reduced paired-pulse inhibition. The loss of inhibition was accompanied by loss of subclasses of inhibitory interneurons labeled by cholecystokinin and the neuronal GABA transporter GAT-1, which project axo-somatic and axo-axonic GABAergic inhibitory terminals to granule cells and axon initial segments. Seizure-induced loss of interneurons providing axo-somatic and axo-axonic inhibition may regulate spike output to pyramidal neurons in CA3 and could play an important role in generation of spontaneous seizures. The sequence of progressive cellular alterations induced by repeated seizures, particularly loss of GABAergic interneurons providing axo-somatic and axo-axonic inhibition, may be important in the development of intractable epilepsy. (+info)
Residues in the extracellular loop 4 are critical for maintaining the conformational equilibrium of the gamma-aminobutyric acid transporter-1.
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We mutated residues Met345 and Thr349 in the rat gamma-aminobutyric acid transporter-1 (GAT-1) to histidines (M345H and T349H). These two residues are located four amino acids apart at the extracellular end of transmembrane segment 7 in a region of GAT-1 that we have previously suggested undergoes conformational changes critical for the transport process. The two single mutants and the double mutant (M345H/T349H) were expressed in Xenopus laevis oocytes, and their steady-state and presteady-state kinetics were examined and compared with wild type GAT-1 by using the two-electrode voltage clamp method. Oocytes expressing M345H showed a decrease in apparent GABA affinity, an increase in apparent affinity for Na+, a shift in the charge/voltage (Q/Vm) relationship to more positive membrane potentials, and an increased Li+-induced leak current. Oocytes expressing T349H showed an increase in apparent GABA affinity, a decrease in apparent Na+ affinity, a profound shift in the Q/Vm relationship to more negative potentials, and a decreased Li+-induced leak current. The data are consistent with a shift in the conformational equilibrium of the mutant transporters, with M345H stabilized in an outward-facing conformation and T349H in an inward-facing conformation. These data suggest that the extracellular end of transmembrane domain 7 not only undergoes conformational changes critical for the translocation process but also plays a role in regulating the conformational equilibrium between inward- and outward-facing conformations. (+info)
GABA transporter-1 (GAT1)-deficient mice: differential tonic activation of GABAA versus GABAB receptors in the hippocampus.
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After its release from interneurons in the CNS, the major inhibitory neurotransmitter GABA is taken up by GABA transporters (GATs). The predominant neuronal GABA transporter GAT1 is localized in GABAergic axons and nerve terminals, where it is thought to influence GABAergic synaptic transmission, but the details of this regulation are unclear. To address this issue, we have generated a strain of GAT1-deficient mice. We observed a large increase in a tonic postsynaptic hippocampal GABAA receptor-mediated conductance. There was little or no change in the waveform or amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) or miniature IPSCs. In contrast, the frequency of quantal GABA release was one-third of wild type (WT), although the densities of GABAA receptors, GABAB receptors, glutamic acid decarboxylase 65 kDa, and vesicular GAT were unaltered. The GAT1-deficient mice lacked a presynaptic GABAB receptor tone, present in WT mice, which reduces the frequency of spontaneous IPSCs. We conclude that GAT1 deficiency leads to enhanced extracellular GABA levels resulting in an overactivation of GABAA receptors responsible for a postsynaptic tonic conductance. Chronically elevated GABA levels also downregulate phasic GABA release and reduce presynaptic signaling via GABAB receptors thus causing an enhanced tonic and a diminished phasic inhibition. (+info)