Although synaptic AMPA receptors have been shown to rapidly internalize, synaptic NMDA receptors are reported to be static. It is not certain whether NMDA receptor stability at synaptic sites is an inherent property of the receptor, or is due to stabilization by scaffolding proteins. In this study, we demonstrate that NMDA receptors are internalized in both heterologous cells and neurons, and we define an internalization motif, YEKL, on the distal C-terminus of NR2B. In addition, we show that the synaptic protein PSD-95 inhibits NR2B-mediated internalization, and that deletion of the PDZ-binding domain of NR2B increases internalization in neurons. This suggests an involvement for PSD-95 in NMDA receptor regulation and an explanation for NMDA receptor stability at synaptic sites.

The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. We investigated the contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons. We identified an ER retention signal (RRR) in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels (Zerangue et al., 1999). We found that surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron.

The regulation of AMPA receptors at the postsynaptic membrane is a fundamental component of synaptic plasticity. In the hippocampus, the induction of long-term potentiation increases the delivery of GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is unclear. We investigated the interaction of GluR1 with SAP97, the only PDZ protein known to interact with GluR1. We find that interactions involving SAP97 and GluR1 occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi. In contrast, few synaptic receptors associate with SAP97, suggesting that SAP97 dissociates from the receptor complex at the plasma membrane. We also show that internalization of GluR1, as triggered by NMDAR activation, does not require SAP97. These results implicate GluR1-SAP97 interactions in mechanisms underlying AMPA receptor targeting.

At excitatory synapses, both NMDA and AMPA receptors are localized to the postsynaptic density (PSD). However, unlike AMPA receptors, synaptic NMDA receptors are stable components of the PSD. Even so, surface-expressed NMDA receptors undergo endocytosis, which is more robust early in development and declines during synaptic development. We investigated the subunit-specific contributions to NMDA receptor endocytosis, specifically defining the endocytic motifs and endocytic pathways preferred by the NR2A and NR2B subunits. We find that NR2A and NR2B have distinct endocytic motifs encoded in their distal C termini and that these interact with clathrin adaptor complexes with differing affinities. We also find that NR2A and NR2B sort into different intracellular pathways after endocytosis, with NR2B preferentially trafficking through recycling endosomes. In mature cultures, we find that NR2B undergoes more robust endocytosis than NR2A, consistent with previous studies showing that NR2A is more highly expressed at stable synaptic sites. Our findings demonstrate fundamental differences between NR2A and NR2B that help clarify developmental changes in NMDA receptor trafficking and surface expression.

No abstract

Kainate receptors are a class of ionotropic glutamate receptors that are widely expressed in the mammalian brain, yet little is known about their physiological role or the mechanisms by which they are regulated. Kainate receptors are composed of multiple subunits (GluR5-7; KA1-2), which can combine to form homomeric or heteromeric channels. While the kainate receptor subunit KA2 can combine with GluR5-7 to form heteromeric channels, it does not form functional homomeric channels when expressed alone. In an attempt to identify the molecular mechanisms for this, we have characterized the trafficking and surface expression of KA2. We find that KA2 alone does not traffic to the plasma membrane and is retained in the endoplasmic reticulum (ER). In contrast, co-expression with GluR6 disrupts ER-retention of KA2 and allows plasma membrane expression. Using a chimeric reporter protein we have identified an ER-retention motif within the KA2 cytosolic domain. Recent studies have identified a consensus ER-retention motif (RRR) that is contained within both the NMDA receptor NR1 subunit and K(+) channels. While KA2 contains a similar stretch of amino acids within its C-terminus (RRRRR), unlike the NR1 motif, disruption of this motif with alternating glutamic acid residues does not disrupt ER-retention of KA2, suggesting a unique mechanism regulating KA2 surface expression.

Sea anemones feed by discharging nematocysts into their prey, but the pathway for control of nematocyst discharge is unknown. The purpose of this study was to investigate the ultrastructural evidence of neuro‐nematocyte synapses and to determine the types of synaptic vesicles present at different kinds of nematocyst‐containing cells. The tip and middle of tentacles from small specimens of Aiptasia pallida were prepared for electron microscopy and serial micrographs were examined. We found clear vesicles in synapses on mastigophore‐containing nematocytes and dense‐cored vesicles in synapses on basitrich‐containing nematocytes and on one cnidoblast with a developing nematocyst. In addition, we found reciprocal neuro‐neuronal and sequential neuro‐neuro‐nematocyte synapses in which dense‐cored vesicles were present. It was concluded that : (1) neuro‐nematocyte synapses are present in sea anemones, (2) different kinds of synaptic vesicles are present at cells containing different types of nematocysts, (3) synapses are present on cnidoblasts before the developing nematocyst can be identified and these synapses may have a trophic influence on nematocyst differentiation, and (4) both reciprocal and sequential synapses are present at the nematocyte, suggesting a complex pathway for neural control of nematocyst discharge. J. Morphol. 238:53–62, 1998. © 1998 Wiley‐Liss, Inc.

Using transmission electron microscopy of serially sectioned tentacles from the sea anemone Aiptasia pallida, we located and characterized two types of neuro-spirocyte synapses. Clear vesicles were observed at 10 synapses and dense-cored vesicles at five synapses. The diameters of vesicles at each neuro-spirocyte synapse were averaged; clear vesicles ranged from 49-89 nm in diameter, whereas the dense-cored vesicles ranged from 97-120 nm in diameter. One sequential pair of synapses included a neuro-spirocyte synapse with clear vesicles (81 nm) and a neuro-neuronal synapse with dense-cored vesicles (168 nm). A second synapse on the same cell had dense-cored vesicles (103 nm). An Antho-RFamide-labeled ganglion cell and three different neurites were observed adjacent to spirocytes, but no neuro-spirocyte synapses were present. Many of the spirocytes also were immunoreactive to Antho-RFamide. The presence of sequential neuro-neuro-spirocyte synapses suggests that synaptic modulation may be involved in the neural control of spirocyst discharge. The occurrence of either dense-cored or clear vesicles at neuro-spirocyte synapses suggests that at least two types of neurotransmitter substances control the discharge of spirocysts in sea anemones.