Anne Herb

Heteromeric NMDA receptors: molecular and functional distinction of subtypes

The N-methyl d-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% ientical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate-and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

Pannexins, a family of gap junction proteins expressed in brain

Database search has led to the identification of a family of proteins, the pannexins, which share some structural features with the gap junction forming proteins of invertebrates and vertebrates. The function of these proteins has remained unclear so far. To test the possibility that pannexins underlie electrical communication in the brain, we have investigated their tissue distribution and functional properties. Here, we show that two of these genes, pannexin 1 (Px1) and Px2, are abundantly expressed in the CNS. In many neuronal cell populations, including hippocampus, olfactory bulb, cortex and cerebellum, there is coexpression of both pannexins, whereas in other brain regions, e.g., white matter, only Px1-positive cells were found. On expression in Xenopus oocytes, Px1, but not Px2 forms functional hemichannels. Coinjection of both pannexin RNAs results in hemichannels with functional properties that are different from those formed by Px1 only. In paired oocytes, Px1, alone and in combination with Px2, induces the formation of intercellular channels. The functional characteristics of homomeric Px1 versus heteromeric Px1/Px2 channels and the different expression patterns of Px1 and Px2 in the brain indicate that pannexins form cell type-specific gap junctions with distinct properties that may subserve different functions.

The KA−2 subunit of excitatory amino acid receptors shows widespread expression in brain and forms ion channels with distantly−related subunits

A new ionotropic glutamate receptor subunit termed KA-2, cloned from rat brain cDNA, exhibits high affinity for [3H]kainate (KD approximately 15 nM). KA-2 mRNA is widely expressed in embryonic and adult brain. Homomeric KA-2 expression does not generate agonist-sensitive channels, but currents are observed when KA-2 is coexpressed with GluR5 or GluR6 subunits. Specifically, coexpression of GluR5(R) and KA-2 produces channel activity, whereas homomeric expression of either subunit does not. Currents through heteromeric GluR5(Q)/KA-2 channels show more rapid desensitization and different current-voltage relations when compared with GluR5(Q) currents. GluR6/KA-2 channels are gated by AMPA, which fails to gate homomeric GluR6 receptor channels. These results suggest possible in vivo partnership relations for high affinity kainate receptors.

The rat delta-1 and delta-2 subunits extend the excitatory amino acid receptor family

We have characterized a second member (delta‐2) of a new class of subunits for the ligand‐gated excitatory amino acid receptor superfamily. The sequence of delta‐2 exhibits an average identity of 25% and 18.5% to the non‐NMDA and NMDA receptor subunits, respectively. The rat delta‐2 gene is expressed predominantly in Purkinje cells of the cerebellum whereas only low levels of delta‐1 transcripts are found in the adult brain. However, delta‐1 gene expression undergoes a pronounced developmental peak, with particularly high mRNA levels in the caudate putamen of late embryonic/early postnatal stages.

Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor α4 subunit

A cDNA of rat brain encoding the GABAA receptor α4 subunit has been cloned. Recombinant receptors composed of α4, β2 and γ2 subunits bind with high affinity the GABA agonist [3H]muscimol and the benzodiazepine ‘alcohol antagonist’ [3H]Ro 15‐4513, but fail to bind benzodiazepine agonists. The α4 subunit is expressed mainly in the thalamus, as assessed by in situ hybridization histochemistry, and may participate in a major population of thalamic GABAA receptors. The α4 mRNA is found at lower levels in cortex and caudate putamen, and is rare in cerebellum.

RED2, a brain specific member of the RNA specific adenosine deaminase family

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.

Pannexins in ischemia-induced neurodegeneration

Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) form large-pore nonselective channels in the plasma membrane of cells and were suggested to play a role in the pathophysiology of cerebral ischemia. To directly test a potential contribution of pannexins in ischemia-related mechanisms, we performed experiments in Px1−/−, Px2−/−, and Px1−/−Px2−/− knockout mice. IL-1β release, channel function in astrocytes, and cortical spreading depolarization were not altered in Px1−/−Px2−/− mice, indicating that, in contrast to previous concepts, these processes occur normally in the absence of pannexin channels. However, ischemia-induced dye release from cortical neurons was lower, indicating that channel function in Px1−/−Px2−/− neurons was impaired. Furthermore, Px1−/−Px2−/− mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. In conclusion, our data demonstrate that Px1 and Px2 underlie channel function in neurons and contribute to ischemic brain damage.

Structural Requirements for RNA Editing in Glutamate Receptor Pre-mRNAs by Recombinant Double-stranded RNA Adenosine Deaminase

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.

Molecular biology of glutamate receptors

The ligand-gated receptors for L-glutamate play a central role in acute neuronal degeneration. Recently cDNAs have been isolated for subunits of several glutamate receptor subtypes. By sequence homology all these subunits clearly belong to one large gene family. Several subfamilies exist and match roughly previously pharmacologically and electrophysiologically defined subtypes of glutamate receptors. Currently four genes (GluR A, B, C and D) are known that code for the AMPA subtypes of glutamate receptors. Recombinant expression of wild type and mutated sequences identified a critical residue in the putative TM2 channel-lining segment that controls Ca2+ ion permeability. The arginine (R) found in GluR B subunits at that position renders AMPA channels impermeable for Ca2+ ions, whereas glutamine (Q) containing GluR A, C and D subunits give rise to Ca2+ permeable channels. RNA editing converts the genomically encoded glutamine codon into the arginine codon found in GluR B cDNAs for the Q/R site. NMDA subtypes of glutamate receptors are formed after coexpression of the NR1 cDNA with a cDNA of the NR2 family. Depending on the member of the NR2 family used, NMDA receptors with different kinetical and pharmacological properties are generated. Common to all channels of these NMDA receptors is a high permeability for Ca2+ ions and a voltage dependent block by Mg2+ ions. All currently known NMDA receptor subunits have an asparagine at the Q/R homologous position. We found that this residue is critical for Mg2+ block and Ca2+ permeability of NMDA receptor channels.

Small N-Terminal Deletion by Splicing in Cerebellar α6 Subunit Abolishes GABAA Receptor Function

Abstract: Sequence variation was found in cDNA coding for the extracellular domain of the rat γ‐aminobutyric acid type A (GABAA) receptor α6 subunit. About 20% of polymerase chain reaction (PCR)‐amplified α6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226–255 as estimated by counting single‐stranded phage plaques hybridized specifically to the short (α6S) and long (wild‐type) forms of the α6 mRNA. Genomic PCR revealed an intron located upstream of the 30‐nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the α6S subunit with the GABAA receptor β2 and γ2 subunits in human embryonic kidney 293 cells, were inactive at binding [3H]muscimol and [3H]Ro 15‐4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA‐induced currents, indistinguishable from those produced by β2γ2 receptors. Therefore, the 10 amino acids encoded by the 30‐nucleotide fragment may be essential for the correct assembly or folding of the α6 subunit‐containing receptors.