Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.

Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-Daspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.F ast excitatory synaptic transmission in the mammalian central nervous system is mediated primarily by ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type and Nmethyl-D-aspartate (NMDA)-type ionotropic glutamate receptors, which are coexpressed at many synapses and subserve distinct physiological functions in synaptic transmission (1-3). Although the vast majority of excitatory synapses in the hippocampus expresses functional NMDA receptors (NMDARs), electrophysiological and anatomical data suggest that the number of AMPA receptors (AMPARs) expressed at individual synapses on CA1 pyramidal cells is highly variable (4-8). Furthermore, recent evidence suggests that the surface expression of AMPARs at individual synapses is not fixed but is dynamically regulated by neuronal activity (2, 9-12). This activity-dependent regulation of the synaptic expression of AMPARs may contribute to the changes in synaptic strength that occur during NMDAR-dependent long-term potentiation and long-term depression (11, 12). Surprisingly, little is known about the detailed molecular mechanisms that regulate the number of AMPARs at excitatory synapses. Previously, we showed a pronounced redistribution of AMPARs away from synaptic sites within minutes after the triggering of long-term depression (12) or pharmacological activation of AMPARs (13). Herein, we show that this process is mediated by dynamin-dependent endocytosis and identify a role of NMDAR activation in promoting AMPAR endocytosis under physiological conditions. Materials and MethodsCell Culture and Immunocytochemistry. Hippocampal cultures were prepared as described (12, 13) and were used for experimentation at 2-3 weeks after plating. Surface AMPARs were stained with an antibody recognizing an extracellular epitope (amino acids 271-285) of the rat GluR1 subunit (Oncogene Research). Before treatment, antibody (5 g͞ml) was applied to live cells for 15 min at 37°C in conditioned cell medium. Cells were then briefly...

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