Hannah Monyer

Developmental and regional expression in the rat brain and functional properties of four NMDA receptors.

An in situ study of mRNAs encoding NMDA receptor subunits in the developing rat CNS revealed that, at all stages, the NR1 gene is expressed in virtually all neurons, whereas the four NR2 transcripts display distinct expression patterns. NR2B and NR2D mRNAs occur prenatally, whereas NR2A and NR2C mRNAs are first detected near birth. All transcripts except NR2D peak around P20. NR2D mRNA, present mainly in midbrain structures, peaks around P7 and thereafter decreases to adult levels. Postnatally, NR2B and NR2C transcript levels change in opposite directions in the cerebellar internal granule cell layer. In the adult hippocampus, NR2A and NR2B mRNAs are prominent in CA1 and CA3 pyramidal cells, but NR2C and NR2D mRNAs occur in different subsets of interneurons. Recombinant binary NR1-NR2 channels show comparable Ca2+ permeabilities, but marked differences in voltage-dependent Mg2+ block and in offset decay time constants. Thus, the distinct expression profiles and functional properties of NR2 subunits provide a basis for NMDA channel heterogeneity in the brain.

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.

Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS

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.

Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit

Functionally diverse GluR channels of the AMPA subtype are generated by the assembly of GluR-A, -B, -C, and -D subunits into homo- and heteromeric channels. The GluR-B subunit is dominant in determining functional properties of heteromeric AMPA receptors. This subunit exists in developmentally distinct edited and unedited forms, GluR-B(R) and GluR-B(Q), which differ in a single amino acid in transmembrane segment TM2 (Q/R site). Homomeric GluR-B(R) channels expressed in 293 cells display a low divalent permeability, whereas homomeric GluR-B(Q) and GluR-D channels exhibit a high divalent permeability. Mutational analysis revealed that both the positive charge and the size of the amino acid side chain located at the Q/R site control the divalent permeability of homomeric channels. Coexpression of Q/R site arginine- and glutamine-containing subunits generates cells with varying divalent permeabilities depending on the amounts of expression vectors used for cell transfection. Intermediate divalent permeabilities were traced to the presence of both divalent permeant homomeric and impermeant heteromeric channels. It is suggested that the positive charge contributed by the arginine of the edited GluR-B(R) subunit determines low divalent permeability in heteromeric GluR channels and that changes in GluR-B(R) expression regulate the AMPA receptor-dependent divalent permeability of a cell.

Structural determinants of ion flow through recombinant glutamate receptor channels

Functional glutamate receptor (GluRs) were transiently expressed in cultured mammalian cells from cloned complementary DNAs encoding GluR-A, -B, -C, or -D polypeptides. The steady-state current-voltage (I-V) relations of glutamate- and kainate-induced currents through homomeric channels fell into two classes: channels composed of either the GluR-A, -C, and -D subunits showed doubly rectifying I-V curves, and channels composed of the GluR-B subunits displayed simple outward rectification. The presence of GluR-B subunits in heteromeric GluRs determined the I-V behavior of the resulting channels. Site-directed mutagenesis identified a single amino acid difference (glutamine to arginine) in the putative transmembrane segment TM2 responsible for subunit-specific I-V relationships. The properties of heteromeric wild-type and mutant GluRs revealed that the dominance of GluR-B is due to the arginine residue in the TM2 region.

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.

Differences in Ca2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression

Fast excitatory synaptic transmission in the CNS is mediated by AMPA-type glutamate receptor (GluR) channels. Heterologous expression suggested that the Ca2+ permeability of these receptors critically depends on the subunit composition. Using patch-clamp techniques in brain slices, we found that the Ca2+ permeability of native AMPA-type GluRs was markedly higher in nonpyramidal (PCa/PK approximately 0.63) than in pyramidal (PCa/PK approximately 0.05) neurons of rat neocortex. Analysis of mRNA in single cells indicated that the relative abundance of GluR-B-specific mRNA was significantly lower in nonpyramidal (GluR-B/GluR-non-B approximately 0.3) than in pyramidal (GluR-B/GluR-non-B approximately 3) cells. This suggests that differences in relative abundance of GluR-B-specific mRNA generate functional diversity of AMPA-type GluRs in neurons with respect to Ca2+ permeability.

Glutamate-operated channels: Developmentally early and mature forms arise by alternative splicing

The expression of two alternative splice variants, Flip and Flop, in mRNAs encoding the four AMPA-selective glutamate receptors (GluR-A, -B, -C, and -D) was studied in the developing brain by in situ hybridization. These receptors are expressed prominently before birth, and patterns of distribution for Flip versions remain largely invariant during postnatal brain development. In contrast, the Flop versions are expressed at low levels prior to postnatal day 8. Around this time, the expression of Flop mRNAs increases throughout the brain, reaching adult levels by postnatal day 14. Thus, receptors carrying the Flop module appear to participate in mature receptor forms.

Impaired Electrical Signaling Disrupts Gamma Frequency Oscillations in Connexin 36-Deficient Mice

Neural processing occurs in parallel in distant cortical areas even for simple perceptual tasks. Associated cognitive binding is believed to occur through the interareal synchronization of rhythmic activity in the gamma (30-80 Hz) range. Such oscillations arise as an emergent property of the neuronal network and require conventional chemical neurotransmission. To test the potential role of gap junction-mediated electrical signaling in this network property, we generated mice lacking connexin 36, the major neuronal connexin. Here we show that the loss of this protein disrupts gamma frequency network oscillations in vitro but leaves high frequency (150 Hz) rhythms, which may involve gap junctions between principal cells (Schmitz et al., 2001), unaffected. Thus, specific connexins differentially deployed throughout cortical networks are likely to regulate different functional aspects of neuronal information processing in the mature brain.

Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes

Several new findings have emphasized the role of neuron‐specific gap junction proteins (connexins) and electrical synapses in processing sensory information and in synchronizing the activity of neuronal networks. We have recently shown that pannexins constitute an additional family of proteins that can form gap junction channels in a heterologous expression system and are also widely expressed in distinct neuronal populations in the brain, where they may represent a novel class of electrical synapses. In this study, we have exploited the hemichannel‐forming properties of pannexins to investigate their sensitivity to well‐known connexin blockers. By combining biochemical and electrophysiological approaches, we report here further evidence for the interaction of pannexin1 (Px1) with Px2 and demonstrate that the pharmacological sensitivity of heteromeric Px1/Px2 is similar to that of homomeric Px1 channels. In contrast to most connexins, both Px1 and Px1/Px2 hemichannels were not gated by external Ca2+. In addition, they exhibited a remarkable sensitivity to blockade by carbenoxolone (with an IC50 of ∼5 μm), whereas flufenamic acid exerted only a modest inhibitory effect. The opposite was true in the case of connexin46 (Cx46), thus indicating that gap junction blockers are able to selectively modulate pannexin and connexin channels.