The molecular and neuronal substrates conferring on clozapine its unique and superior efficacy in the treatment of schizophrenia remain elusive. The interaction of clozapine with many G proteincoupled receptors is well documented but less is known about its biologically active metabolite, N-desmethylclozapine. Recent clinical and preclinical evidences of the antipsychotic activity of the muscarinic agonist xanomeline prompted us to investigate the effects of N-desmethylclozapine on cloned human M1-M5 muscarinic receptors. N-desmethylclozapine preferentially bound to M1 muscarinic receptors with an IC 50 of 55 nM and was a more potent partial agonist (EC50, 115 nM and 50% of acetylcholine response) at this receptor than clozapine. Furthermore, pharmacological and site-directed mutagenesis studies suggested that N-desmethylclozapine preferentially activated M1 receptors by interacting with a site that does not fully overlap with the acetylcholine orthosteric site. As hypofunction of N-methyl-D-aspartate (NMDA) receptordriven neuronal ensembles has been implicated in psychotic disorders, the neuronal activity of N-desmethylclozapine was electrophysiologically investigated in hippocampal rat brain slices. N-desmethylclozapine was shown to dose-dependently potentiate NMDA receptor currents in CA1 pyramidal cells by 53% at 100 nM, an effect largely mediated by activation of muscarinic receptors. Altogether, our observations provide direct evidence that the brain penetrant metabolite N-desmethylclozapine is a potent, allosteric agonist at human M1 receptors and is able to potentiate hippocampal NMDA receptor currents through M1 receptor activation. These observations raise the possibility that N-desmethylclozapine contributes to clozapine's clinical activity in schizophrenics through modulation of both muscarinic and glutamatergic neurotransmission.

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Glycine acts as a necessary coagonist for glutamate at the NMDA receptor (NMDAR) complex by binding to the strychnine-insensitive glycine-B binding site on the NR1 subunit. The fact that glycine is normally found in the brain and spinal cord at concentrations that exceed those required to saturate this site has led to the speculation that glycine normally saturates NMDAR-containing synapses in vivo. However, additional lines of evidence suggest that synaptic glycine may be efficiently regulated in synaptic areas by the glycine transporter type 1 (GlyT1). The recent description of a potent and selective GlyT1 inhibitor (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine [NFPS]) provides a tool for evaluation of the hypothesis that inhibition of GlyT1 may increase synaptic glycine and thereby potentiate NMDAR function in vivo. In the present study, we found that (+)-NFPS demonstrated >10-fold greater activity in an in vitro functional glycine reuptake assay relative to the racemic compound. In vivo, (+/-)-NFPS significantly enhanced long-term potentiation in the hippocampal dentate gyrus induced by high-frequency electrical stimulation of the afferent perforant pathway. Furthermore, (+)-NFPS induced a pattern of c-Fos immunoreactivity comparable with the atypical antipsychotic clozapine and enhanced prepulse inhibition of the acoustic startle response in DBA/2J mice, a strain with low basal levels of prepulse inhibition. Collectively, these data suggest that selective inhibition of GlyT1 can enhance NMDAR-sensitive activity in vivo and also support the idea that GlyT1 may represent a novel target for developing therapeutics to treat disorders associated with NMDAR hypofunction.

In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encode a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA.The electrical excitability of cells of the nervous system is regulated, in part, by voltage-sensitive K+ channels (1). The structure of neuronal K+ channels that have been cloned has been conserved from Drosophila to humans and consists of proteins containing six hydrophobic, putative transmembrane domains, one of which, S4, has been implicated in sensing changes in the membrane potential (2-4). Although they are structurally similar, these K+ channels can be divided into four distinct classes by virtue of their sequence homology to channels encoded at the Drosophila Shaker, Shab, Shal, and Shaw loci (5-11). In Drosophila, diversity of the K+ channels within a given class arises, in part, from differential splicing of RNA encoding the channels (5-11). In contrast, mammalian K+ channel genes that have been described to date have no introns in the coding region (12)(13)(14) and the diversity of these channels appears to have resulted from gene duplication and divergence. We describe here, however, the cloning and expression of a cDNA encoding a mammalian Shaw-type K+ channel, Kv4, which arises from alternative splicing of a transcript from a gene that also encodes NGK2 (15), another mammalian channel related to Shaw. 11 MATERIALS AND METHODS K,4 cDNA Clones. An oligo(dT)-primed cDNA library, constructed from mRNA isolated from the brains of 2-weekold rats, was screened using a probe prepared from clone K41 (12), encoding part of a Shaker-type channel, labeled to a specific activity of >1 x 109 by random hexamer-primed labeling (17). The filters were hybridized at 60'C in 5 x SSPE (lx SSPE = 0.18 M NaCl/10 mM sodium phosphate, pH 7.4/1 mM EDTA), 5x Denhardt's solution, 0.5% SDS, and 1% glycerol with 200 ,ug of salmon sperm DNA per ml and probe at 1 x 106 cpm/ml and were washed to a final stringency of 0.2x SSPE/0.1% SDS at 220C. A cDNA (SA) containing a 2.6-kilobase (kb) insert was isolated and subcloned into pBS+ for sequencing. To obtain the 3' end of the coding region, another oligo(dT)-primed cDNA library, constructed from mRNA isolated from the brains of 14-day-old rats, was screened using an oligonucleotide probe (0123) that included bases 1260-1359 from 5A (Fig. 1). The probe was labeled to a specific activity of >1 x 108 cpm/pmol by filling in two overlapping oligonucleotides with the Klenow fragment of DNA polymerase I and all four [a-32P]dNTPs. Filters were hybridized at 4...