Interactions of alkali cations with glutamate transporters

The transport of glutamate is coupled to the co-transport of three Na+ ions and the countertransport of one K+ ion. In addition to this carrier-type exchange behaviour, glutamate transporters also behave as chloride channels. The chloride channel activity is strongly influenced by the cations that are involved in coupled flux, making glutamate transporters representative of the ambiguous interface between carriers and channels. In this paper, we review the interaction of alkali cations with glutamate transporters in terms of these diverse functions. We also present a model derived from electrostatic mapping of the predicted cation-binding sites in the X-ray crystal structure of the Pyrococcus horikoshii transporter GltPh and in its human glutamate transporter homologue EAAT3. Two predicted Na+-binding sites were found to overlap precisely with the Tl+ densities observed in the aspartate-bound complex. A novel third site predicted to favourably bind Na+ (but not Tl+) is formed by interaction with the substrate and the occluding HP2 loop. A fourth predicted site in the apo state exhibits selectivity for K+ over both Na+ and Tl+. Notably, this K+ site partially overlaps the glutamate-binding site, and their binding is mutually exclusive. These results are consistent with kinetic and structural data and suggest a plausible mechanism for the flux coupling of glutamate with Na+ and K+ ions

Interactions of alkali cations with glutamate transporters

The transport of glutamate is coupled to the co-transport of three Na+ ions and the countertransport of one K+ ion. In addition to this carrier-type exchange behaviour, glutamate transporters also behave as chloride channels. The chloride channel activity is strongly influenced by the cations that are involved in coupled flux, making glutamate transporters representative of the ambiguous interface between carriers and channels. In this paper, we review the interaction of alkali cations with glutamate transporters in terms of these diverse functions. We also present a model derived from electrostatic mapping of the predicted cation-binding sites in the X-ray crystal structure of the Pyrococcus horikoshii transporter GltPh and in its human glutamate transporter homologue EAAT3. Two predicted Na+-binding sites were found to overlap precisely with the Tl+ densities observed in the aspartate-bound complex. A novel third site predicted to favourably bind Na+ (but not Tl+) is formed by interaction with the substrate and the occluding HP2 loop. A fourth predicted site in the apo state exhibits selectivity for K+ over both Na+ and Tl+. Notably, this K+ site partially overlaps the glutamate-binding site, and their binding is mutually exclusive. These results are consistent with kinetic and structural data and suggest a plausible mechanism for the flux coupling of glutamate with Na+ and K+ ions

Specificity and Actions of an Arylaspartate Inhibitor of Glutamate Transport at the Schaffer Collateral-CA1 Pyramidal Cell Synapse

In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg2+ concentrations, but in Mg2+-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg2+ was the same in hippocampal slices from EAAT3 +/+ and EAAT3 −/− mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg2+ revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg2+ block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling

Differences between patients with ventricular tachycardia and ventricular fibrillation as assessed by signal-averaged electrocardiogram, radionuclide ventriculography and cardiac mapping

AbstractThis study examined 65 patients with ventricular tachycardia or fibrillation late after myocardial infarction to determine whether they differed with respect to duration of ventricular activation in sinus rhythm and left ventricular ejection fraction. Patients with spontaneous ventricular tachycardia had a longer ventricular activation time in sinus rhythm than did patients with spontaneous ventricular fibrillation. This difference was detected with the signal-averaged electrocardiogram (ECG) (tachycardia 181 ± 33 ms, fibrillation 152 ± 23 ms, p < 0.001) and at epicardial mapping (tachycardia 210 ± 17 ms, fibrillation 192 ± 17 ms, p < 0.02). Left ventricular ejection fraction was lower in patients with spontaneous ventricular tachycardia (0.22 ± 0.09) than in patients with spontaneous ventricular fibrillation (0.27 ± 0.09) (p < 0.05).The patients with both spontaneous and inducible ventricular fibrillation had a shorter ventricular activation time on the signal-averaged ECG (129 ± 17 ms) and a higher ejection fraction (0.36 ± 0.05) than did either patients with spontaneous ventricular fibrillation and inducible ventricular tachycardia (158 ± 21 ms and 0.25 ± 0.08, respectively, each p < 0.01) or patients with both spontaneous and inducible ventricular tachycardia (181 ± 33 ms and 0.22 ± 0.09, respectively, each p < 0.001). Of the patients with inducible ventricular tachycardia, presentation with tachycardia rather than fibrillation was associated with a longer ventricular activation time on the signal-averaged ECG (181 ± 33 versus 158 ± 21 ms, p < 0.02) and a longer cycle length of inducible ventricular tachycardia (290 ± 61 versus 259 ± 44 ms, p = 0.05).In conclusion, conduction delay during sinus rhythm and left ventricular dysfunction appear to be greatest in patients with spontaneous and inducible ventricular tachycardia, and least in patients with spontaneous and inducible ventricular fibrillation

Specificity and Actions of an Arylaspartate Inhibitor of Glutamate Transport at the Schaffer Collateral-CA1 Pyramidal Cell Synapse

In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg2+ concentrations, but in Mg2+-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg2+ was the same in hippocampal slices from EAAT3 +/+ and EAAT3 −/− mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg2+ revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg2+ block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling

The Fourteenth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the extended Baryon Oscillation Spectroscopic Survey and from the second phase of the Apache Point Observatory Galactic Evolution Experiment

The fourth generation of the Sloan Digital Sky Survey (SDSS-IV) has been in operation since July 2014. This paper describes the second data release from this phase, and the fourteenth from SDSS overall (making this, Data Release Fourteen or DR14). This release makes public data taken by SDSS-IV in its first two years of operation (July 2014-2016). Like all previous SDSS releases, DR14 is cumulative, including the most recent reductions and calibrations of all data taken by SDSS since the first phase began operations in 2000. New in DR14 is the first public release of data from the extended Baryon Oscillation Spectroscopic Survey (eBOSS); the first data from the second phase of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE-2), including stellar parameter estimates from an innovative data driven machine learning algorithm known as "The Cannon"; and almost twice as many data cubes from the Mapping Nearby Galaxies at APO (MaNGA) survey as were in the previous release (N = 2812 in total). This paper describes the location and format of the publicly available data from SDSS-IV surveys. We provide references to the important technical papers describing how these data have been taken (both targeting and observation details) and processed for scientific use. The SDSS website (www.sdss.org) has been updated for this release, and provides links to data downloads, as well as tutorials and examples of data use. SDSS-IV is planning to continue to collect astronomical data until 2020, and will be followed by SDSS-V.Comment: SDSS-IV collaboration alphabetical author data release paper. DR14 happened on 31st July 2017. 19 pages, 5 figures. Accepted by ApJS on 28th Nov 2017 (this is the "post-print" and "post-proofs" version; minor corrections only from v1, and most of errors found in proofs corrected

Mouse DRG Cell Line with Properties of Nociceptors

In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons