Identification of domains and amino acids involved in GLuR7 ion channel function.

The kainate receptors GluR6 and GluR7 differ considerably in their ion channel properties, despite sharing 86% amino acid sequence identity. When expressed in Xenopus oocytes GluR6 conducts large agonist-evoked currents, whereas GluR7 lacks measurable currents. In the present study, we localized the determinants that are responsible for the functional differences between GluR6 and GluR7 to the extracellular loop domain L3. In addition, we generated several GluR7 point mutants that are able to conduct currents that can be readily measured in Xenopus oocytes. In GluR6, glutamate- and kainate-evoked maximal currents are of the same magnitude when desensitization is inhibited with the lectin concanavalin A. By contrast, all functional GluR7 mutants were found to have glutamate current amplitudes significantly larger than those evoked by kainate. We localized the domain that determines the relative agonist efficacies to the C-terminal half of the L3 domain of GluR7. Our data show that EC(50) values for glutamate (but not for kainate) in GluR7 mutants or chimeras tend to be increased in comparison to the EC(50) values in GluR6. The high EC(50) for wild-type GluR7 reported in the literature appears to be linked to the S1 portion of the agonist-binding domain. Finally, we determined the C-terminal half of the L3 domain plus the far C-terminal domain of GluR7 to be responsible for the recently reported reduction of current amplitude seen when GluR7 is coexpressed with GluR6. We conclude that coexpression of GluR6 and GluR7 leads to nonstochastical assembly of heteromeric receptor complexes.

Mutant cycle analysis of the active and desensitized states of an AMPA receptor induced by willardiines.

The halogenated willardiines are agonists at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptors. Although they differ only by the nature of the halogen substituent, they display marked differences in their efficacy to activate the receptor channel opening and in causing desensitization. We have studied the origin of the different agonist properties of the willardiines and in particular the nature of the structural element within the receptor binding domain that is able to distinguish between willardiines at a subatomic resolution of 0.6 A (the difference in radius between F and Br) and allow (S)-5-fluorowillardiine to cause receptor desensitization much more than (S)-5-bromowillardiine. For this purpose, we analyzed, with the thermodynamic mutant cycle method, the active and desensitized states induced by the willardiines in the GluR1 subtype of AMPA receptors and GluR1 mutants in which residues E398, Y446, L646, and S650, within the agonist binding domain, were mutated. The results were used to generate a 3D model of the willardiine docking mode. We suggest that the active and desensitized states of the AMPA-R correspond, respectively, to the open-lobe and closed-lobe conformations of the agonist binding domain.

Purification procedure and monoclonal antibodies: two instruments for research on vertebrate porins.

On Western blots of skeletal muscle preparations of different vertebrate classes, four monoclonal anti-human type 1 porin antibodies recognize one single band of either 30.5 or 31 kDa, respectively. To confirm that it is eukaryotic porin which is labeled by the antibodies, we used a purification procedure developed for human type 1 porin for porins from skeletal muscle of shark, frog, and turkey. Applied to different mammalian species and tissues, this procedure exclusively provides type 1 porin. However, applied to shark skeletal muscle, it provides two porin isotypes in nearly equal amounts. In the case of frog skeletal muscle, the procedure provides mainly type 2 porin and a lower amount of type 1 porin. Applied to turkey skeletal muscle, the method provides exclusively type 2 porin. As demonstrated by two-dimensional Western blots, both shark and frog porin isotypes and the turkey type 2 porin are recognized by our antibodies. Furthermore, we elucidated the entire amino acid sequence of frog type 2 porin.

Investigation by ion channel domain transplantation of rat glutamate receptor subunits, orphan receptors and a putative NMDA receptor subunit.

Among the 18 ionotropic glutamate receptor subunits identified in the mammalian central nervous system, five (delta1, delta2, GluR7, chi2 and NR3A, formerly called NMDAR-L or chi1) reportedly fail to form functional ion channels in heterologous expression systems. Four of these subunits, delta1, delta2, chi2 and NR3A, have not even been shown to bind glutamatergic ligands, relegating them to the status of 'orphan' receptors. We used a domain transplantation approach to investigate potential functional properties of the putative ion channel domains of four of these subunits. By exchanging ion pore domains between functional glutamate receptors (GluR1, GluR6 and NMDAR1) with known pore properties we first tested the feasibility of the domain swapping method. We demonstrate that ion channel domains can be transplanted between all three functional subfamilies of ionotropic glutamate receptors. Furthermore, exchange of ion pore domains allows identification of those channel properties determined exclusively by the ion pore. We then show that transplanting the pore domain of GluR7 into either GluR1 or GluR6 generates perfectly functional ligand-gated ion channels that allow characterization of electrophysiological and pharmacological properties of the GluR7 pore domain. In contrast, delta1, delta2 and NR3A do not produce functional receptors when their pore domains are transplanted into either the AMPA receptor, GluR1, the kainate receptor, GluR6, or the NMDA receptor, NMDAR1. We speculate that the orphan receptors delta1 and delta2, and the NMDA receptor-like subunit NR3A may serve some modulatory function, rather than contributing to the formation of ion channels.

A desensitization-inhibiting mutation in the glutamate binding site of rat alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunits is dominant in heteromultimeric complexes.

Recently, it has been shown that a single leucine-to-tyrosine mutation in the agonist binding domains of the homomerically expressed alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors GluR3 and GluR1 is sufficient to completely block receptor desensitization. In the present study we tested heteromeric subunit combinations of AMPA receptors to demonstrate that the block of desensitization afforded by this mutation is dominant in heteromeric subunit complexes containing the leucine-to-tyrosine mutation in at least one of the subunits. In addition, by comparing mutated, desensitization-deficient forms of unedited GluR1 and GluR1 edited at the Q/R-site of the ion pore we demonstrate that the desensitization properties of AMPA receptors are not linked to the editing state of the ion pore domain and thus are independent of the permeability properties of the ion channel.

Thermoterrabacterium ferrireducens gen. nov., sp. nov., a thermophilic anaerobic dissimilatory Fe(III)-reducing bacterium from a continental hot spring.

A strain of a thermophilic, anaerobic, dissimilatory, Fe(III)-reducing bacterium, Thermoterrabacterium ferrireducens gen. nov., sp. nov. (type strain JW/AS-Y7T; DSM 11255), was isolated from hot springs in Yellowstone National Park and New Zealand. The gram-positive-staining cells occurred singly or in pairs as straight to slightly curved rods, 0.3 to 0.4 by 1.6 to 2.7 microns, with rounded ends and exhibited a tumbling motility. Spores were not observed. The temperature range for growth was 50 to 74 degrees C with an optimum at 65 degrees C. The pH range for growth at 65 degrees C was from 5.5 to 7.6, with an optimum at 6.0 to 6.2. The organism coupled the oxidation of glycerol to reduction of amorphous Fe(III) oxide or Fe(III) citrate as an electron acceptor. In the presence as well as in the absence of Fe(III) and in the presence of CO2, glycerol was metabolized by incomplete oxidation to acetate as the only organic metabolic product; no H2 was produced during growth. The organism utilized glycerol, lactate, 1,2-propanediol, glycerate, pyruvate, glucose, fructose, mannose, and yeast extract as substrates. In the presence of Fe(III) the bacterium utilized molecular hydrogen. The organism reduced 9,10-anthraquinone-2,6-disulfonic acid, fumarate (to succinate), and thiosulfate (to elemental sulfur) but did not reduce MnO2, nitrate, sulfate, sulfite, or elemental sulfur. The G + C content of the DNA was 41 mol% (as determined by high-performance liquid chromatography). The 16S ribosomal DNA sequence analysis placed the isolated strain as a member of a new genus within the gram-type-positive Bacillus-Clostridium subphylum.