Abstract:The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in Xenopus oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses. Key Words: Postsynaptic density protein PSD-95-Src-Insulin-Protein kinase C-NMDA receptorXenopus oocyte. J. Neurochem. 75, 282-287 (2000).Glutamate receptors at synapses interact with a raft of proteins involved in signaling. The NMDA subtype interacts with a particular conglomerate of signaling proteins including the postsynaptic density protein PSD-95. PSD-95 is attached to the NR2 subunit C terminus via the last three amino acid residues (S/TXV) but does not interact with NR1 (Kornau et al., 1995;Niethammer et al., 1996;Sheng and Pak, 1999). PSD-95 in turn interacts with other potential signaling molecules such as a neuronal RasGTPase-activating protein and neuronal nitric oxide synthase (Sheng and Pak, 1999). The signal transduction role of PSD-95 was first shown in gene knockout mice that have a lowered threshold for longterm potentiation induction and impaired long-term depression and spatial learning (Migaud et al., 1998). Although Ca 2ϩ entering through the NMDA receptor (NMDAR) and activating either calcium/calmodulin-dependent kinase II or calcineurin is critical for the induction of long-term potentiation/depression, modulation of NMDA currents by other kinases, including Src, may also be required for long-term potentiation (Lu et al., 1998;Huang and Hsu, 1999;Yu and Salter, 1999). Recently, protein kinase C (PKC)-mediated modulation of NMDA currents and Fyn modulation of NMDAR phosphorylation state have been found to be altered by the receptor-associated protein PSD-95. PSD-95 interferes with PKC potentiation of the mouse NR2B/NR1 current in Xenopus oocytes (Yamada et al., 1999). PSD-95 also accentuates NR2A phosphorylation by the Src family protein tyrosine kinase (PTK) Fyn (Tezuka et al., 1999).Therefore, in the present study, we tested the possibility that PSD-95 would have an opposite, facilitatory effect on Src potentiation of NR2A/N...

Self-referencing H+-selective microelectrodes were used to measure extracellular proton fluxes from cone-driven horizontal cells isolated from the retina of the catfish (Ictalurus punctatus). The neurotransmitter glutamate induced an alkalinization of the area adjacent to the external face of the cell membrane. The effect of glutamate occurred regardless of whether the external solution was buffered with 1 mM HEPES, 3 mM phosphate, or 24 mM bicarbonate. The AMPA/kainate receptor agonist kainate and the NMDA receptor agonist N-methyl-d-aspartate both mimicked the effect of glutamate. The effect of kainate on proton flux was inhibited by the AMPA/kainate receptor blocker CNQX, and the effect of NMDA was abolished by the NMDA receptor antagonist DAP-5. Metabotropic glutamate receptor agonists produced no alteration in proton fluxes from horizontal cells. Depolarization of cells either by increasing extracellular potassium or directly by voltage clamp also produced an alkalinization adjacent to the cell membrane. The effects of depolarization on proton flux were blocked by 10 μM nifedipine, an inhibitor of L-type calcium channels. The plasmalemma Ca2+/H+ ATPase (PMCA) blocker 5(6)-carboxyeosin also significantly reduced proton flux modulation by glutamate. Our results are consistent with the hypothesis that glutamate-induced extracellular alkalinizations arise from activation of the PMCA pump following increased intracellular calcium entry into cells. This process might help to relieve suppression of photoreceptor neurotransmitter release that results from exocytosed protons from photoreceptor synaptic terminals. Our findings argue strongly against the hypothesis that protons released by horizontal cells act as the inhibitory feedback neurotransmitter that creates the surround portion of the receptive fields of retinal neurons.

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 μm) and SKF89976‐A (100 μm), but was unaffected by 100 μm picrotoxin. Prior application of 100 μm 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.

Extracellular H(+) has been hypothesized to mediate feedback inhibition from horizontal cells onto vertebrate photoreceptors. According to this hypothesis, depolarization of horizontal cells should induce extracellular acidification adjacent to the cell membrane. Experiments testing this hypothesis have produced conflicting results. Studies examining carp and goldfish horizontal cells loaded with the pH-sensitive dye 5-hexadecanoylaminofluorescein (HAF) reported an extracellular acidification on depolarization by glutamate or potassium. However, investigations using H(+)-selective microelectrodes report an extracellular alkalinization on depolarization of skate and catfish horizontal cells. These studies differed in the species and extracellular pH buffer used and the presence or absence of cobalt. We used both techniques to examine H(+) changes from isolated catfish horizontal cells under identical experimental conditions (1 mM HEPES, no cobalt). HAF fluorescence indicated an acidification response to high extracellular potassium or glutamate. However, a clear extracellular alkalinization was found using H(+)-selective microelectrodes under the same conditions. Confocal microscopy revealed that HAF was not localized exclusively to the extracellular surface, but rather was detected throughout the intracellular compartment. A high degree of colocalization between HAF and the mitochondrion-specific dye MitoTracker was observed. When HAF fluorescence was monitored from optical sections from the center of a cell, glutamate produced an intracellular acidification. These results are consistent with a model in which depolarization allows calcium influx, followed by activation of a Ca(2+)/H(+) plasma membrane ATPase. Our results suggest that HAF is reporting intracellular pH changes and that depolarization of horizontal cells induces an extracellular alkalinization, which may relieve H(+)-mediated inhibition of photoreceptor synaptic transmission.

The H(+) hypothesis of lateral feedback inhibition in the outer retina predicts that depolarizing agents should increase H(+) release from horizontal cells. To test this hypothesis, self-referencing H(+) -selective microelectrodes were used to measure extracellular H(+) fluxes from isolated goldfish horizontal cells. We found a more complex pattern of cellular responses than previously observed from horizontal cells of other species examined using this technique. One class of cells had an initial standing signal indicative of high extracellular H(+) adjacent to the cell membrane; challenge with glutamate, kainate or high extracellular potassium induced an extracellular alkalinization. This alkalinization was reduced by the calcium channel blockers nifedipine and cobalt. A second class of cells displayed spontaneous oscillations in extracellular H(+) that were abolished by cobalt, nifedipine and low extracellular calcium. A strong correlation between changes in intracellular calcium and extracellular proton flux was detected in experiments simultaneously monitoring intracellular calcium and extracellular H(+) . A third set of cells was characterized by a standing extracellular alkalinization which was turned into an acidic signal by cobalt. In this last set of cells, addition of glutamate or high extracellular potassium did not significantly alter the proton signal. Taken together, the response characteristics of all three sets of neurons are most parsimoniously explained by activation of a plasma membrane Ca(2+) ATPase pump, with an extracellular alkalinization resulting from exchange of intracellular calcium for extracellular H(+) . These findings argue strongly against the hypothesis that H(+) release from horizontal cells mediates lateral inhibition in the outer retina.