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R E Oswald

Publications and source records attributed to R E Oswald.

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

Three-dimensional models of non-NMDA glutamate receptors.

Structural models have been produced for three types of non-NMDA inotropic glutamate receptors: an AMPA receptor, GluR1, a kainate receptor, GluR6; and a low-molecular-weight kainate receptor from goldfish, GFKAR alpha. Modeling was restricted to the domains of the proteins that bind the neurotransmitter glutamate and that form the ion channel. Model building combined homology modeling, distance geometry, molecular mechanics, interactive modeling, and known constraints. The models indicate new potential interactions in the extracellular domain between protein and agonists, and suggest that the transition from the "closed" to the "open" state involves the movement of a conserved positive residue away from, and two conserved negative residues into, the extracellular entrance to the pore upon binding. As a first approximation, the ion channel domain was modeled with a structure comprising a central antiparallel beta-barrel that partially crosses the membrane, and against which alpha-helices from each subunit are packed; a third alpha-helix packs against these two helices in each subunit. Much, but not all, of the available data were consistent with this structure. Modifying the beta-barrel to a loop-like topology produced a model consistent with available data.

Amino Acid Sequence↗
PubMed1996

Ligand-binding characteristics and related structural features of the expressed goldfish kainate receptors: identification of a conserved disulfide bond and three residues important for ligand binding.

Low-molecular-weight kainate receptors from nonmammalian vertebrate brain belong structurally to the ionotropic glutamate receptor superfamily. In this study, two previously cloned goldfish kainate receptor subunits (GFKAR alpha and GFKAR beta) were transiently expressed in human embryonic kidney 293 cells, and their ligand-binding properties and some associated structural features were characterized, resulting in the following findings. 1) Both subunits form homomeric receptors with high affinity for [3H]kainate (KD = 16 and 31 nM, respectively) and L-glutamate (KD = 2 and 40 microM, respectively). 2) A deletion mutant lacking the originally proposed second-transmembrane domain was efficiently expressed and retains the overall ligand-binding properties of wild-type GFKAR alpha, strongly indicating that this region is not a transmembrane domain. 3) Mutations of Q12, A53, and Y54 of GFKAR beta indicate that these three residues are important for ligand binding (particularly L-glutamate), which is consistent with the sequence homology to bacterial periplasmic binding proteins. 4) Mutation of the three extracellular cysteine residues of GFKAR beta indicated that the two conserved cysteine residues (C305 and C385), located between two transmembrane segments, form a solvent-accessible disulfide bond. Analysis of [3H]kainate binding to wild-type and cysteine mutations of GFKAR beta indicate that in the absence of the disulfide bond, the affinity for kainate is increased 3-fold. These data lend further evidence in support of a model of glutamate receptor topology with three transmembrane segments and reveal several general structural features of the extracellular ligand-binding domain of the kainate receptors. These results are consistent with the notion that the ligand-binding domain has close structural similarities to bacterial periplasmic binding proteins.

Amino Acid Sequence↗
PubMed1995

Asn-265 of frog kainate binding protein is a functional glycosylation site: implications for the transmembrane topology of glutamate receptors.

Kainate binding proteins (KBPs) from frog and goldfish brain are glycosylated, integral membrane proteins. These KBPs are homologous (35-40%) to the C-terminal half of AMPA and kainate receptors which have been shown to form glutamate-gated ion channels. We report here that the frog KBP has three functional N-glycosylation sites. Of particular interest, Asn-265, a residue located between two putative membrane spanning regions of the frog KBP, is a functional N-glycosylation site. A mutation of Ser-267 to Gly renders this site non-functional as shown using an in vitro translation system and by transient expression in human embryonic kidney (HEK 293) cells. The mutant receptor protein (S267G), when expressed in HEK cells, binds kainate with high affinity (Kd = 16 nM). These results further support a topology with three transmembrane segments for KBPs and, by sequence homology, for glutamate-gated ion channels.

Amino Acid Sequence↗
PubMed1995

A topological analysis of goldfish kainate receptors predicts three transmembrane segments.

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in vertebrate brain. We have previously cloned cDNAs encoding two homologous kainate receptors (GFKAR alpha, 45 kDa, and GFKAR beta, 41 kDa) from goldfish brain and proposed a topology with three transmembrane domains (Wo, Z. G., and Oswald, R. E. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7154-7158). These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific antibodies and point and deletion mutations. We report here that the entire region between the previously proposed third and fourth transmembrane segments is translocated and likely to be extracellular in mature receptors. This was based on the following results. 1) The entire segment was protected from Proteinase K and trypsin digestion and could be immunoprecipitated by a site-specific antibody. 2) Functional sites for N-glycosylation are present in the C-terminal half of the segment, and 3) a mutation, constructed with an additional consensus site for N-glycosylation in the N-terminal half of the segment, was found to be glycosylated at that site. Given the fact that the N terminus of the protein is likely to be extracellular, this would place an even number of transmembrane segments between the extracellular N terminus and the glycosylated segment. In addition, results of N-glycosylation and proteolysis protection assays of GFKAR alpha mutations indicated that the previously proposed second transmembrane segment is not a true transmembrane domain. These results provide further evidence in support of a topology with three transmembrane domains that has important implications for the relationship of structure to function in ionotropic glutamate receptors.

Amino Acid Sequence↗
PubMed1995

Linear prediction and single-channel recording.

The measurement of individual single-channel events arising from the gating of ion channels provides a detailed data set from which the kinetic mechanism of a channel can be deduced. In many cases, the pattern of dwells in the open and closed states is very complex, and the kinetic mechanism and parameters are not easily determined. Assuming a Markov model for channel kinetics, the probability density function for open and closed time dwells should consist of a sum of decaying exponentials. One method of approaching the kinetic analysis of such a system is to determine the number of exponentials and the corresponding parameters which comprise the open and closed dwell time distributions. These can then be compared to the relaxations predicted from the kinetic model to determine, where possible, the kinetic constants. We report here the use of a linear technique, linear prediction/singular value decomposition, to determine the number of exponentials and the exponential parameters. Using simulated distributions and comparing with standard maximum-likelihood analysis, the singular value decomposition techniques provide advantages in some situations and are a useful adjunct to other single-channel analysis techniques.

Algorithms↗
PubMed1995

Unraveling the modular design of glutamate-gated ion channels.

Glutamate receptors that function as ligand-gated ion channels are essential components of cell-cell communication in the nervous system. Despite a wealth of information concerning these receptors, details of their structure are just beginning to emerge. We propose that glutamate receptors comprise four modules: two modules that are related to bacterial periplasmic-binding proteins, one module that is related to the pore-forming region of K+ channels, and one regulatory module of unknown origin. A K(+)-channel-like domain inserted into a crucial region of a periplasmic-binding protein-like domain suggests a mechanism for transduction of binding energy to channel opening. This modular design also suggests an evolutionary link between a ligand-gated ion-channel family and voltage-gated ion channels.

Amino Acid Sequence↗
PubMed1994

Transmembrane topology of two kainate receptor subunits revealed by N-glycosylation.

Glutamate receptors are the primary excitatory neurotransmitter receptors in vertebrate brain and are of critical importance to a wide variety of neurological processes. Recent reports suggest that ionotropic glutamate receptors may have a unique transmembrane topology not shared by other ligand-gated ion channels. We report here the cloning of cDNAs from goldfish brain encoding two homologous kainate receptors with protein molecular masses of 41 kDa. Using a cell-free translation/translocation system, we show that (i) a portion of these receptors previously thought to be a large intracellular loop is actually located extracellularly and (ii) the putative second transmembrane region of the receptor thought to line the ion channel may not be a true membrane-spanning domain. An alternative model for the transmembrane topology of kainate receptors is proposed that could potentially serve as a framework for future detailed study of the structure of this important class of neurotransmitter receptors.

Amino Acid Sequence↗
PubMed1994

Computer simulations of cyclic and acyclic cholinergic agonists: conformational search and molecular dynamics simulations.

Molecular dynamics simulations have been performed on aqueous solutions of two chemically similar nicotinic cholinergic agonists in order to compare their structural and dynamical differences. The cyclic 1,1-dimethyl-4-acetylpiperazinium iodide (HPIP) molecule was previously shown to be a strong agonist for nicotinic acetylcholine receptors (McGroddy et al., 1993), while the acyclic N,N,N,N'-tetramethyl-N'-acetylethylenediamine iodide (HTED) derivative is much less potent. These differences were expected to arise from differences in the solution structures and internal dynamics of the two molecules. HPIP was originally thought to be relatively rigid; however, molecular dynamics simulations suggest that the acetyl portion of the molecule undergoes significant ring dynamics on a psec timescale. The less constrained HTED molecule is relatively rigid, with only one transition observed about any of the major dihedrals in four 100 psec simulations, each started from a different conformation. The average structures obtained from the simulations are very similar to the starting minimized structure in each case, except for the HTED simulation where a single rotation about the N-C-C-N(+) backbone occurred. In each case, HTED had three to five more water molecules in its primary solvation shell than HPIP, indicating that differences in the energetics of desolvation before binding may partially explain the increased potency of HPIP as compared to HTED.

Biophysical Phenomena↗
PubMed1994

The beta subunit of neuronal nicotinic acetylcholine receptors is a determinant of the affinity for substance P inhibition.

Substance P is known to inhibit nicotinic acetylcholine receptors from neuronal tissue, skeletal muscle, and electroplaque. The interaction of substance P with specific combinations of neuronal nicotinic acetylcholine receptor subunits was studied by expressing various combinations of subunits in Xenopus oocytes. The response to acetylcholine was inhibited by substance P with all subunit combinations tested; however, the apparent affinity for substance P varied by 20-30-fold. The affinity seemed to be dependent on the beta subtype expressed (beta 4 or beta 2). This suggests that the beta subunit may contribute, at least partially, to the substance P binding site. In the case of the alpha 7 subtype, which forms a homooligomeric receptor, the apparent affinity for substance P was intermediate between those of the two beta subtypes coexpressed with either alpha 3 or alpha 4. As previously found, the inhibition was noncompetitive. Furthermore, the inhibition was not voltage dependent and, therefore, is unlikely to be due to substance P blocking the channel within the transmembrane portion of the pore.

Acetylcholine↗
PubMed1993

Channel blocking properties of a series of nicotinic cholinergic agonists.

Inhibition of the nicotinic acetylcholine receptor (nAChR) by channel blockade has been demonstrated with a variety of large organic cations, including several nicotinic agonists. We have studied the kinetics of channel blocking of a series of agonists which vary systematically in size and hydrophobicity due to a hydrocarbon chain from one to six carbons in length, as well as one agonist with a tertiary isomer of one hydrocarbon chain. Single-channel recording was used in combination with three different analysis techniques for determining the kinetic and equilibrium parameters of channel blockade. With an increasing number of methylenes, the blocking rates were essentially constant and the unblocking rates decreased exponentially. This is consistent with studies of the blocking properties of alcohols at the nAChR channel. Also, a linear decrease in the depth to which the larger agonists penetrate the membrane spanning region of the channel was observed. The three smaller agonists, however, all traverse approximately 75% of the membrane field, in agreement with previous measurements of the location of the narrowest region of the channel, the selectivity filter.

Animals↗
PubMed1993

Solution structure and dynamics of cyclic and acyclic cholinergic agonists.

Two classes of nicotinic cholinergic agonists, which vary in flexibility and electronegativity, have been synthesized, and their structural and dynamic properties have been studied with nuclear magnetic resonance (NMR) spectroscopy. Although the compounds are chemically identical except for the presence or absence of one cyclicizing C--C bond, single channel recording and radioligand binding studies have shown that the cyclic compounds are considerably more potent than the acyclic derivatives (McGroddy, K.A., A.A. Carter, M.M. Tubbert, and R.E. Oswald. 1993. Biophys. J. 64:325-338). Using one- and two-dimensional NMR spectroscopy, we have shown that these molecules exist in two distinct stable conformers, which differ in the orientation of the amide bond. The cyclic 1,1-dimethyl-4-trifluoroacetyl-piperazinium iodide and its trifluoromethyl derivative compounds are symmetric, and the two conformers are of equal energy. The acyclic N,N,N,N'-tetramethyl-N'-acetylethylene-diamine iodide (TED) and its trifluoromethyl derivative derivatives, however, populate two energetically unequal solution conformations. Using variable temperature NMR spectroscopy on these molecules and their uncharged precursors, we have characterized the energetics of amide bond isomerization and have distinguished steric and electrostatic contributions to the equilibrium between the two conformers. The more populated TED conformer has the amide methyl group trans to the carbonyl oxygen, and it is stabilized by an electrostatic attraction between the partially negative carbonyl oxygen and the positively charged quaternary amine nitrogen. As discussed in the accompanying paper (McGroddy, K.A., A.A. Carter, M.M. Tubbert, and R.E. Oswald. 1993. Biophys. J. 64:325-338), the differences in the stable solution structures of the TED derivatives and their interconversion kinetics may be of biological significance.

Animals↗
PubMed1993

Analysis of cyclic and acyclic nicotinic cholinergic agonists using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy.

The relationship between the structure and function of a series of nicotinic cholinergic agonists has been studied using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy. The cyclic compound 1,1-dimethyl-4-acetylpiperazinium iodide and its trifluoromethyl analogue (F3-PIP) interact with nicotinic acetylcholine receptors (nAChRs) from both Torpedo electroplaque and BC3H-1 cells at lower concentrations than the acyclic derivatives, N,N,N,N'-tetramethyl-N'-acetylethylenediamine iodide and its fluorinated analogue (F3-TED). The magnitude of the difference in potencies depends on the type of measurement. In binding experiments, the differences between the two classes of compounds depends mainly on the conditions of the experiment. In measurements of the initial interaction with the nAChR, the PIP compounds have an affinity approximately one order of magnitude higher than that of the TED compounds. Longer incubations indicated that the PIP compounds were able to induce a time-dependent shift in receptor affinity consistent with desensitization, whereas the TED compounds were unable to induce such a shift. The activation of single channel currents by the cyclic compounds occurs at concentrations approximately two orders of magnitude lower than for the acyclic compounds, but the TED compounds exhibit a larger degree of channel blockade than the PIP compounds. Previous work (McGroddy, K.A., and R.E. Oswald. 1992. Biophys. J. 64:314-324) has shown that the TED compounds can exist in two energetically distinct conformational states related by an isomerization of the amide bond. 19F nuclear magnetic resonance experiments suggest that the higher energy population of the TED compounds may interact preferentially with the ACh binding sites on the nAChRs and that a significant fraction of the difference between the initial affinity of the PIP and TED compounds may be accounted for by the predominance in solution of a conformational state less able to interact with the ACh binding sites on nAChRs.

Animals↗
PubMed1993

Identification of the benzomorphan opiate binding site on the catalytic subunit of acetylcholinesterase.

The interaction of a benzomorphan opiate with the active site of the catalytic subunit of acetylcholinesterase was studied using photoaffinity labeling. UV irradiation of (-)-N-[3H]allylnormetazocine bound to Torpedo acetylcholinesterase resulted in covalent incorporation of 60-70% of the bound ligand. The labeled catalytic subunit was subjected to chemical cleavage with cyanogen bromide and proteolytic degradation with trypsin, chymotrypsin, and staphylococcal V8 protease. The resulting peptide fragments were purified by high performance liquid chromatography and sequenced in the gas phase. The label was not stable under the conditions of the sequencing, but a peptide fragment consisting of Gln74 to Glu82 was reproducibly labeled. These amino acids are located at the rim of a gorge leading to the active site of the enzyme. Molecular modeling studies then demonstrated that these residues can be placed within van der Waals contact of the (-)-N-[3H]allylnormetazocine molecule while it is bound to the active site of the enzyme.

Acetylcholinesterase↗
PubMed1992

Interaction of the frog brain kainate receptor expressed in Chinese hamster ovary cells with a GTP-binding protein.

A protein that binds kainate with high affinity has been purified and cloned from frog brain (Rana pipiens) and has approximately 35% sequence homology with mammalian non-N-methyl-D-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. Frog brain membranes and membranes from Chinese hamster ovary (CHO) cells transfected with the cDNA coding for the frog kainate-binding protein (CHO-4 cells) bound kainate with essentially identical affinity (KD values of 1.9 and 2.1 nM, respectively). In both tissues, the affinity for kainate decreased 9-fold in the presence of 100 microM GTP gamma S (guanosine 5'-O-(3-thio)triphosphate). No specific kainate binding to nontransfected CHO cell membranes was observed. GTP gamma S and GDP were effective inhibitors of kainate binding, while cGMP and adenosine 5'-O-(3-thio)triphosphate had no effect in either frog brain membranes or CHO-4 membranes. Pretreatment of CHO-4 cell membranes with pertussis toxin led to a 34% decrease in kainate binding. Kainate increased the binding of [3H]5'-guanylyl imidodiphosphate by 61%, and the rate of GTP hydrolysis by up to 5-fold. These results indicate that the kainate receptor cloned from frog brain can interact functionally with a G protein present in CHO-4 cell membranes.

Adenosine Triphosphate↗