Recording of glutamate-activated currents in membrane patches was combined with RT-PCR-mediated AMPA receptor (AMPAR) subunit mRNA analysis in single identified cells of rat brain slices. Analysis of AMPARs in principal neurons and interneurons of hippocampus and neocortex and in auditory relay neurons and Bergmann glial cells indicates that the GluR-B subunit in its flip version determines formation of receptors with relatively slow gating, whereas the GluR-D subunit promotes assembly of more rapidly gated receptors. The relation between Ca2+ permeability of AMPAR channels and the relative GluR-B mRNA abundance is consistent with the dominance of this subunit in determining the Ca2+ permeability of native receptors. The results suggest that differential expression of GluR-B and GluR-D subunit genes, as well as splicing and editing of their mRNAs, account for the differences in gating and Ca2+ permeability of native AMPAR channels.

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor channels mediate the fast component of excitatory postsynaptic currents in the central nervous system. Site-selective nuclear RNA editing controls the calcium permeability of these channels, and RNA editing at a second site is shown here to affect the kinetic aspects of these channels in rat brain. In three of the four AMPA receptor subunits (GluR-B, -C, and -D), intronic elements determine a codon switch (AGA, arginine, to GGA, glycine) in the primary transcripts in a position termed the R/G site, which immediately precedes the alternatively spliced modules "flip" and "flop." The extent of editing at this site progresses with brain development in a manner specific for subunit and splice form, and edited channels possess faster recovery rates from desensitization.

Editing of RNA by site-selective adenosine deamination alters codons in brain-expressed pre-messenger RNAs for glutamate receptor (GluR) subunits including a codon for a channel determinant (Q/R site) in GluR-B, which controls the Ca2+ permeability of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. Editing of GluR pre-mRNAs requires a double-stranded RNA (dsRNA) structure formed by exonic and intronic sequences and is catalysed by an unknown dsRNA adenosine deaminase. Here we report the cloning of complementary DNA for RED1, a dsRNA adenosine deaminase expressed in brain and peripheral tissues that efficiently edits the Q/R site in GluR-B pre-mRNA in vitro. This site is poorly edited by DRADA, which is distantly sequence-related to RED1. Both deaminases edit the R/G site in GluR-B pre-mRNA, indicating that members of an emerging gene family catalyse adenosine deamination in nuclear transcripts with distinct but overlapping substrate specificities.

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.

The mammalian RNA-specific adenosine deaminases DRADA/dsRAD (alias ADAR) and RED1 (alias ADARB1) have been implicated in the site-selective editing of brain-expressed pre-mRNAs for glutamate receptor subunits and of antigenomic RNA of hepatitis delta virus. These enzymes are expressed in many if not all tissues, predicting an as yet unappreciated significance for adenosine deamination-mediated recoding of gene transcripts in the mammalian organism. We now report the molecular cloning of cDNA for RED2 (alias ADARB2), a third member of the RNA-specific adenosine deaminase family in the rodent. RED2 is closely sequence-related to RED1 but appears to be expressed only in the brain, where expression is widespread reaching highest levels in olfactory bulb and thalamus. RED2 further differs from RED1 in having a 54-residue amino-terminal extension which includes an arginine-rich motif. Different from DRADA and RED1, recombinantly expressed RED2 did not deaminate adenosines in extended synthetic dsRNA or in GluR-B pre-mRNA. However, a chimera of RED1 and RED2 edited the GluR-B Q/R and R/G sites with moderate efficiency. Our data suggest that RED2 may edit brain-specific transcripts with distinct structural features.RNA editing by nucleotide conversion occurs in some mammalian gene transcripts, altering the RNA's informational capacity (reviewed in Ref. 1). A cytosine to uridine deamination is observed in the transcript for intestinal apolipoprotein B (apoB 1 ; reviewed in Ref.2), and adenosine deamination occurs in pre-mRNAs for subunits of glutamate-gated receptor channels (GluR) mediating excitatory synaptic transmission in the central nervous system (reviewed in Refs. 3 and 4). Whereas apoB editing involves linear sequence recognition, GluR pre-mRNA editing requires in vitro (5-7) and in vivo (8) the formation of a double-stranded (ds) RNA configured from exonic and intronic sequences. This requirement for a dsRNA structure and the observed conversion of adenosine to inosine in particular GluR pre-mRNA codons (9 -11) predicts catalysis by dsRNA-specific adenosine deaminases. Indeed, molecular cloning has revealed the existence of two dsRNA adenosine deaminases, expressed in many if not all tissues. These enzymes, termed DRADA (dsRNA adenosine deaminase; also termed dsRAD or ADAR) (12-14) and RED1 (dsRNA-specific editase 1 or ADARB1; Ref. 15), share a common domain architecture with several dsRNA binding domains (dsRBDs; Ref. 16) being located amino-terminal to a catalytic deamination domain. This latter domain is defined by conserved amino acid residues containing the putative Zn 2ϩ -chelating and protontransferring residues necessary for adenosine deamination (13,15,17). These are also found in other nucleoside deaminases (18), including the cytidine deaminase APOBEC1 (apoB editing catalytic component 1) for apoB editing (19). Recombinantly expressed DRADA and RED1 deaminate in vitro up to 50% of the adenosines in extended, synthetic dsRNA, used as an artificial substrate (15, 20 -23). Furthermore, DRADA edits the R...

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Pre-mRNAs for brain-expressed ionotropic glutamate receptor subunits undergo RNA editing by site-specific adenosine deamination, which alters codons for molecular determinants of channel function. This nuclear process requires double-stranded RNA structures formed by exonic and intronic sequences in the pre-mRNA and is likely to be catalyzed by an adenosine deaminase that recognizes these structures as a substrate. DRADA, a double-stranded RNA adenosine deaminase, is a candidate enzyme for L-glutamate-activated receptor channel (GluR) pre-mRNA editing. We show here that DRADA indeed edits GluR pre-mRNAs, but that it displays selectivity for certain editing sites. Recombinantly expressed DRADA, both in its full-length form and in an N-terminally truncated version, edited the Q/R site in GluR6 pre-mRNA and the R/G site but not the Q/R site of GluR-B pre-mRNA. This substrate selectivity correlated with the base pairing status and sequence environment of the editing-targeted adenosines. The Q/R site of GluR-B pre-mRNA was edited by an activity partially purified from HeLa cells and thus differently structured editing sites in GluR pre-mRNAs appear to be substrates for different enzymatic activities.The alteration of codons by RNA editing, leading to changes in protein structure and function, represents a newly recognized type of posttranscriptional modification in mammalian nuclear transcripts and occurs by site-specific base modification (1, 2). In the transcript for intestinal apolipoprotein B (apoB) 1 , a translational stop codon is generated by cytidine deamination, generating the expression of a truncated protein with altered function (1). By contrast, specific adenosines are deaminated (2, 3) in pre-mRNAs for subunits of glutamategated receptor channels (GluR) (4). At the Q/R site (5) of the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR-B and the high-affinity kainate receptor subunits GluR5 and GluR6, a glutamine codon CAG is converted to the arginine codon CIG. At the R/G site of the AMPA receptor subunits GluR-B, -C, and -D (6) an arginine codon AGA is switched to the glycine codon IGA, and at the I/V and Y/C sites in GluR6, an ATT codon is changed to ITT and a TAC to TIC, respectively (7). Each of the amino acid changes generated by RNA editing alters functional properties of the glutamate-activated channel (3, 4).Different from apoB RNA editing (1), the site-specific adenosine deamination in GluR transcripts requires a doublestranded (ds)RNA structure formed by the exonic sequence around the editing site and an intronic editing site complementary sequence (ECS) (3,8), predicting that this type of RNA editing is catalyzed by an adenosine deaminase that operates on dsRNA. In addition to exonic adenosines, some intronic adenosines are also converted, including hotspot1 in GluR-B intron 11 (8) and in GluR6 pre-mRNA (9). The site-selective adenosine to inosine conversion in GluR-B pre-mRNA could be demonstrated in vitro (10 -12). dsRAD (2, 13), also termed DRADA (14), is a d...

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