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N Burnashev

Publications and source records attributed to N Burnashev.

At least 37 records · Page 2Linked to original sources
PubMed1996

Calcium permeability of glutamate-gated channels in the central nervous system.

Molecular cloning of ionotropic glutamate receptors and the development of new measurement techniques have significantly advanced our understanding of the molecular mechanisms controlling ligand-mediated entry of Ca2+ into neurons of the mammalian CNS. Recent studies have demonstrated that various types of glutamate receptors expressed in different nerve cells are permeable to Ca2+ to variable extents, depending on the structural peculiarities of the subunits and their composition in a particular cell. This diversity provides a regulable pathway for Ca2+ entry during synaptic transmission. The fractional contribution of this Ca2+ to the total synaptic current might be a substantial means of elevating the intracellular Ca2+ concentration over a wide temporal range.

Animals↗
PubMed1995

Block of native Ca(2+)-permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification.

1. The influence of intracellular factors on current rectification of different subtypes of native alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate receptors (AMPARs) was studied in rat brain slices by combining fast application of glutamate with patch pipette perfusion. 2. The peak current-voltage (I-V) relation of the AMPARs expressed in Bergmann glial cells of cerebellum and dentate gyrus (DG) basket cells of hippocampus was weakly rectifying in outside-out patches and nystatin-perforated vesicles, but showed a doubly rectifying shape with a region of reduced slope between 0 and +40 mV in nucleated patches. The I-V relation of AMPARs expressed in hippocampal CA3 pyramidal neurones was linear in all recording configurations. 3. Intracellular application of 25 microM spermine, a naturally occurring polyamine, blocked outward currents in outside-out patches from Bergmann glial cells and DG basket cells in a voltage-dependent manner, generating I-V relations with a doubly rectifying shape which were similar to those recorded in nucleated patches. AMPARs in CA3 pyramidal cell patches were unaffected by 25 microM spermine. 4. The half-maximal blocking concentration of spermine at +40 mV was 0.3 microM in Bergmann glial cell patches and 1.5 microM in DG basket cell patches, whereas it was much higher (>> 100 microM) for CA3 pyramidal cell patches. Spermidine also affected current rectification, but with lower affinity. The block of outward current by polyamines following voltage jumps developed within < 0.5 ms. 5. We conclude that current rectification, rather than being an intrinsic property of the Ca(2+)-permeable AMPAR channel, is generated by polyamine block.

Animals↗
PubMed1995

Fractional calcium currents through recombinant GluR channels of the NMDA, AMPA and kainate receptor subtypes.

1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye fura-2 were made in HEK 293 cells expressing recombinant glutamate receptor (GluR) channels, and fractional Ca2+ currents (the proportion of whole-cell current carried by Ca2+; Pf) were determined. 2. Cells expressing N-methyl-D-aspartate receptor (NMDAR) channels showed glutamate activated Ca2+ inflow in the voltage range -60 to 40 mV in normal extracellular solution. Ca2+ inflow decreased in a voltage-dependent manner at membrane potentials more negative than -30 mV due to Mg2+ block. Voltage dependence of block at negative potentials was stronger in cells expressing the NR1-NR2A as compared with cells expressing NR1-NR2C subunits. 3. Fractional Ca2+ currents through NMDARs were independent of extracellular Mg2+ and varied between 8.2% (NR1-NR2C subunits) and 11% (NR1-NR2A subunits) in normal extracellular solution (1.8 mM Ca2+) at -60 mV membrane potential. Pf values increased with increasing [Ca2+]o in the range of 0.5-10mM [Ca2+]o in a saturating fashion. 4. In cells expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits which were unedited at the Q/R site of the putative transmembrane segment TM2 (Q-form), or in cells coexpressing unedited and edited subunits (R-form), the glutamate-evoked Ca2+ inflow increased from 20 to -80 mV in an almost linear way. 5. Fractional Ca2+ currents through AMPAR channels depended on subunit composition. Pf values of Q-form homomeric channels at -60 mV and 1.8 mM [Ca2+]o were between 3.2 and 3.9%. They were slightly voltage dependent and increased with [Ca2+]o in the range 1.8-10mM. Pf values in cells co-expressing Q- and R-form subunits were almost one order of magnitude smaller (0.54%). 6. Relative concentrations of Q-form and R-form GluR-B subunit-specific cDNAs used for cell transfection determined the expression of functionally different heteromeric AMPARS. Pf decreased with increasing relative concentration of R-form encoding CDNAs from 3.4 to 1.4%, demonstrating that editing of the Q/R site of GluR-B subunits decreases Ca2+ inflow through heteromeric AMPARs. 7. Cells expressing the GluR-6 subunit of the kainate receptor (KAR) family were characterized by Pf values which depended on the editing in the TM1 and TM2 segments. Pf values were largest for the Q-form (1.55-2.0%) and lowest for R-form channels (< 0.2%), suggesting that Q/R site editing also decreases Ca2+ inflow through KAR channels. Cells co-expressing both subunit forms showed an intermediate value (0.58%).(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium Channel Agonists↗
PubMed1995

Dimensions of the narrow portion of a recombinant NMDA receptor channel.

Glutamate-activated single-channel and ensemble currents were recorded from Xenopus laevis oocytes and HEK 293 cells expressing a recombinant NMDA receptor, assembled from NR1 and NR2A subunits. Cesium was the main charge carrier, and organic cations were used to determine the presence of vestibules of this channel and to estimate its pore diameter. The large organic cations tris-(hydroxymethyl)-aminomethane (Tris), N-methyl-glucamine (NMG), arginine (NMG), arginine (Arg), choline, and tetramethylammonium (TMA), when added in millimolar concentrations to the extracellular or cytoplasmic side, produced a voltage-dependent blockade of single-channel Cs+ currents. These molecules behaved as impermeant ions that only partially traverse the channel from either side. The smaller cations trimethylammonium (TriMA) and dimethylammonium (DMA) produced a small and nearly voltage-independent reduction in current amplitude, suggesting that they are permeant. In biionic experiments with Cs+ as the reference ion, the large blocking cations NMG, Arg, Tris, TMA, choline, hexamethonium (Hme), triethylammonium (TriEA), and tetraethylammonium (TEA) showed no measurable permeability. TriMA and smaller ammonium derivatives were permeant. Both the permeability and single-channel conductance of organic cations, relative to Cs+, decreased as the ion size increased. The results suggest that the NMDA receptor has extracellular and cytoplasmic mouths that can accommodate large cations up to 7.3 A in mean diameter. The narrow portion of the pore is estimated to have a mean diameter of 5.5 A.

Animals↗
PubMed1994

A molecular determinant for submillisecond desensitization in glutamate receptors.

The decay of excitatory postsynaptic currents in central neurons mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors is likely to be shaped either by receptor desensitization or by offset after removal of glutamate from the synaptic cleft. Native AMPA receptors show desensitization time constants of 1 to about 10 milliseconds, but the underlying molecular determinants of these large differences are unknown. Cloned AMPA receptors carrying the "flop" splice variants of glutamate receptor subtype C (GluR-C) and GluR-D are shown to have desensitization time constants of around 1 millisecond, whereas those with the "flip" variants are about four times slower. Cerebellar granule cells switch their expression of GluR-D splice variants from mostly flip forms in early stages to predominantly flop forms in the adult rat brain. These findings suggest that rapid desensitization of AMPA receptors can be regulated by the expression and alternative splicing of GluR-D gene transcripts.

Alternative Splicing↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

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.

Animals↗
PubMed1993

Determinants of Ca2+ permeability in both TM1 and TM2 of high affinity kainate receptor channels: diversity by RNA editing.

GluR6, a subunit of high affinity kainate receptor channels in the mammalian CNS, carries a glutamine (Q) or arginine (R) residue in a critical position (Q/R site) of the putative channel-forming segment TM2. One form, GluR6(Q), is encoded by the GluR6 gene; the other, GluR6(R), is generated by RNA editing. Further analysis of cloned GluR6 cDNA revealed that two additional positions, located in transmembrane segment TM1, are diversified by RNA editing to generate either isoleucine (I) or valine (V) in one and tyrosine (Y) or cysteine (C) in the other TM1 position. In GluR6 channels, in contrast with AMPA receptor channels, the presence of Q in the TM2 Q/R site determines channels with low Ca2+ permeability, whereas an R determines a higher Ca2+ permeability if TM1 is fully edited. In the TM1 unedited form of GluR6, Ca2+ permeability is less dependent on the presence of either Q or R in TM2. Thus Ca2+ permeability of kainate receptor channels can vary, depending on editing of both TM1 and TM2.

Amino Acid Sequence↗
PubMed1992

Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor.

The N-methyl-D-aspartate (NMDA) receptor forms a cation-selective channel with a high calcium permeability and sensitivity to channel block by extracellular magnesium. These properties, which are believed to be important for the induction of long-term changes in synaptic strength, are imparted by asparagine residues in a putative channel-forming segment of the protein, transmembrane 2 (TM2). In the NR1 subunit, replacement of this asparagine by a glutamine residue decreases calcium permeability of the channel and slightly reduces magnesium block. The same substitution in NR2 subunits strongly reduces magnesium block and increases the magnesium permeability but barely affects calcium permeability. These asparagines are in a position homologous to the site in the TM2 region (Q/R site) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that is occupied by either glutamine (Q) or arginine (R) and that controls divalent cation permeability of the AMPA receptor channel. Hence AMPA and NMDA receptor channels contain common structural motifs in their TM2 segments that are responsible for some of their ion selectivity and conductance properties.

Amino Acid Sequence↗
PubMed1992

Calcium-permeable AMPA-kainate receptors in fusiform cerebellar glial cells.

Glutamate-operated ion channels (GluR channels) of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-kainate subtype are found in both neurons and glial cells of the central nervous system. These channels are assembled from the GluR-A, -B, -C, and -D subunits; channels containing a GluR-B subunit show an outwardly rectifying current-voltage relation and low calcium permeability, whereas channels lacking the GluR-B subunit are characterized by a doubly rectifying current-voltage relation and high calcium permeability. Most cell types in the central nervous system coexpress several subunits, including GluR-B. However, Bergmann glia in rat cerebellum do not express GluR-B subunit genes. In a subset of cultured cerebellar glial cells, likely derived from Bergmann glial cells. GluR channels exhibit doubly rectifying current-voltage relations and high calcium permeability, whereas GluR channels of cerebellar neurons have low calcium permeability. Thus, differential expression of the GluR-B subunit gene in neurons and glia is one mechanism by which functional properties of native GluR channels are regulated.

Animals↗
PubMed1992

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% identical 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.

Amino Acid Sequence↗
PubMed1992

A glutamate receptor channel with high affinity for domoate and kainate.

The non-NMDA family of glutamate receptors comprises a growing number of structurally related subunits (GluR-A to -D or -1 to -4; GluR-5, -6; KA-1). GluR-A to -D appear to constitute the major AMPA receptor subtypes but the functional and pharmacological characteristics of the other subunits are unresolved. Using a mammalian expression system we demonstrate here that homomeric GluR-5 receptors exhibit properties of a high affinity domoate (KD approximately 2 nM) and kainate (KD approximately 70 nM) binding site. For these receptors, the rank order of ligands competing with [3H]kainate binding was domoate much greater than quisqualate approximately glutamate much greater than AMPA approximately CNQX. The respective receptor channels were gated in decreasing order of sensitivity by domoate, kainate, glutamate and AMPA. In contrast to recombinantly expressed GluR-A to -D channels, currents elicited at GluR-5 receptor desensitize channels to all agonists. This property is characteristic of currents in peripheral neurons on sensory ganglia. These findings suggest the existence of at least two distinct types of non-NMDA receptor channels, both gated by AMPA and kainate, but differing in pharmacology and current properties.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Base Sequence↗
PubMed1992

Glutamate-gated ion channels in the brain. Genetic mechanism for generating molecular and functional diversity.

L-glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Most cells are responsive to glutamate which activates cation channels with different pharmacological, kinetic, and ion permeability properties. These channels play important roles in neurotransmission, memory acquisition as well as acute and chronic disorders of the brain. The present report summarizes recent knowledge on AMPA (a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) receptors which mediate fast synaptic neurotransmission.

Animals↗
PubMed1991

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.

Amino Acid Sequence↗