Transmitter Binding Sites Research Articles

Mapping the conformational wave of acetylcholine receptor channel gating.

Allosteric transitions allow fast regulation of protein function in living systems. Even though the end points of such conformational changes are known for many proteins, the characteristics of the paths connecting these states remain largely unexplored. Rate-equilibrium linear free-energy relationships (LFERs) provide information about such pathways by relating changes in the free energy of the transition state to those of the ground states upon systematic perturbation of the system. Here we present an LFER analysis of the gating reaction pathway of the muscle acetylcholine receptor. We studied the closed <==> open conformational change at the single-molecule level following perturbation by series of single-site mutations, agonists and membrane voltages. This method provided a snapshot of several regions of the receptor at the transition state in terms of their approximate positions along the reaction coordinate, on a scale from 0 (closed-like) to 1 (open-like). The resulting map reveals a spatial gradient of positional values, which suggests that the conformational change proceeds in a wave-like manner, with the low-to-high affinity change at the transmitter-binding sites preceding the complete opening of the pore.

Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists.

1. The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis. 2. The dissociation equilibrium constants were (mM): ACh (0. 16)<oxotremorine M (0.6)<carbamylcholine (0.8)<nicotine (2.6). 3. The gating equilibrium constants (opening/closing) were: ACh (45)>carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0. 5)>muscarine (0.15). 4. Rat neuronal alpha4beta2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined. 5. Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site.

Strychnine activates neuronal alpha7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains.

Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric alpha7 receptors, and that mutating Leu-247 of the alpha7 nicotinic AcCho receptor-channel domain (L247Talpha7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type alpha7 or L247Talpha7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Talpha(7) receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189-190Salpha7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker alpha-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic alpha7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical alpha7 subunits may be sufficient to determine the functional properties of alpha7 receptors.

A re‐examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics

1 During routine sequencing of our mouse muscle α subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305-7 (residue 433, in the M4 domain) the database lists GTC which encodes a valine, while our putative ‘wild-type’ cDNA had the nucleotides GCC, which encodes an alanine. No other sequence differences were found. 2 PCR amplification of genomic DNA confirmed that the BALB/C mouse α subunit gene has a T nucleotide at position 1306, and, therefore, that the protein has a V at position 433 in the M4 segment. 3 In order to determine the functional consequences of this difference, either wild-type (V433) or mutant (A433) α subunits were co-expressed in HEK cells with mouse β, ε and δ subunits. Single-channel currents were recorded in cell-attached patches, and rate and equilibrium constants were estimated from open and closed durations obtained from a range of ACh concentrations. No significant differences were found between the activation rate constants or equilibrium constants of the V433 and A433 variants. 4 Kinetic modelling of αV433 AChR suggests that the two transmitter binding sites have similar dissociation equilibrium constants for acetylcholine (∼160 μM, in 142 mM extracellular KCl). 5 Diliganded AChRs occupy a closed state that has a lifetime of ∼1 ms. The rate constants for entering and leaving this state do not vary with the ACh concentration. 6 The kinetics of a mutant AChR that causes a slow channel congenital myaesthenic syndrome, αG153S, was re-examined. The properties of this mutant were similar with a V or an A at position α433.

A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics.

1. During routine sequencing of our mouse muscle alpha subunit acetylcholine receptor channel (AChR) cDNA clones, we detected a discrepancy with the GenBank database entry (accession X03986). At nucleotides 1305-7 (residue 433, in the M4 domain) the database lists GTC which encodes a valine, while our putative 'wild-type' cDNA had the nucleotides GCC, which encodes an alanine. No other sequence differences were found. 2. PCR amplification of genomic DNA confirmed that the BALB/C mouse alpha subunit gene has a T nucleotide at position 1306, and, therefore, that the protein has a V at position 433 in the M4 segment. 3. In order to determine the functional consequences of this difference, either wild-type (V433) or mutant (A433) alpha subunits were co-expressed in HEK cells with mouse beta, epsilon and delta subunits. Single-channel currents were recorded in cell-attached patches, and rate and equilibrium constants were estimated from open and closed durations obtained from a range of ACh concentrations. No significant differences were found between the activation rate constants or equilibrium constants of the V433 and A433 variants. 4. Kinetic modelling of alphaV433 AChR suggests that the two transmitter binding sites have similar dissociation equilibrium constants for acetylcholine ( approximately 160 microM in 142 mM extracellular KCl). 5. Diliganded AChRs occupy a closed state that has a lifetime of approximately 1 ms. The rate constants for entering and leaving this state do not vary with the ACh concentration. 6. The kinetics of a mutant AChR that causes a slow channel congenital myaesthenic syndrome, alphaG153S, was re-examined. The properties of this mutant were similar with a V or an A at position alpha433.

Desensitization of mouse nicotinic acetylcholine receptor channels. A two-gate mechanism.

The rate constants of acetylcholine receptor channels (AChR) desensitization and recovery were estimated from the durations and frequencies of clusters of single-channel currents. Diliganded-open AChR desensitize much faster than either unliganded- or diliganded-closed AChR, which indicates that the desensitization rate constant depends on the status of the activation gate rather than the occupancy of the transmitter binding sites. The desensitization rate constant does not change with the nature of the agonist, the membrane potential, the species of permeant cation, channel block by ACh, the subunit composition (epsilon or gamma), or several mutations that are near the transmitter binding sites. The results are discussed in terms of cyclic models of AChR activation, desensitization, and recovery. In particular, a mechanism by which activation and desensitization are mediated by two distinct, but interrelated, gates in the ion permeation pathway is proposed.

The Somatosensory Cortex of Human: Cytoarchitecture and Regional Distributions of Receptor-Binding Sites

The aim of this study is to characterize the regional and laminar distribution patterns of various neurotransmitter binding sites in areas 3a, 3b, 1, and 2 of the human primary somatosensory cortex, and to compare these receptor-based “maps” with the cytoarchitectonic parcelation. Cryostat sections from a dorsomedial region of the postcentral gyrus close to the interhemispheric fissure and from a ventrolateral region close to the Sylvian fissure were examined. Neurotransmitter-binding sites were analyzed with quantitativein vitroreceptor autoradiography. Different muscarinic-binding sites were labeled with [3H]pirenzepine and [3H]oxotremorine-M, noradrenergic-binding sites with [3H]prazosin, different serotoninergic-binding sites with [3H]5-hydroxytryptamine and [3H]ketanserine, glutamate-binding sites withl-[3H]glutamate, and GABA-binding sites with [3H]muscimol. Adjacent sections were stained with a modified Nissl method for cytoarchitectonic analysis. The binding sites either were preferentially localized in the superficial layers ([3H]5-hydroxytryptamine, [3H]prazosin,l-[3H]glutamate, [3H]muscimol, and [3H]pirenzepine) or were more homogeneously distributed with highest densities in layers III–V ([3H]oxotremorine-M and [3H]ketanserine). Changes in the distribution patterns of [3H]oxotremorine-M- and [3H]ketanserine-binding sites precisely matched the borders between areas 4/3a, 3b/1, and 1/2, as defined cytoarchitectonically. In addition, the autoradiographs showed that area 1 possibly consists of two subregions which cannot be distinguished cytoarchitectonically. The results demonstrate that the regional and laminar distribution patterns of some, but not all, transmitter-binding sites are precisely correlated with the cytoarchitectonic parcelation of the human primary somatosensory cortex. In addition, binding sites may reveal new borders not detectable in Nissl-stained sections. Finally, the human primary somatosensory cortex differs clearly from the primary motor cortex due to higher densities ofl-[3H]glutamate-, [3H]muscimol-, [3H]pirenzepine-, [3H]oxotremorine-M-, and [3H]ketanserine-binding sites.

Two different areas within the primary motor cortex of man.

The primary motor area (M1) of mammals has long been considered to be structurally and functionally homogeneous. This area corresponds to Brodmann's cytoarchitectural area 4. A few reports showing that arm and hand are doubly represented in M1 of macaque monkeys and perhaps man, and that each subarea has separate connections from somatosensory areas, have, with a few exceptions, gone largely unnoticed. Here we show that area 4 in man can be subdivided into areas '4 anterior' (4a) and '4 posterior' (4p) on the basis of both quantitative cytoarchitecture and quantitative distributions of transmitter-binding sites. We also show by positron emission tomography that two representations of the fingers exist, one in area 4a and one in area 4p. Roughness discrimination activated area 4p significantly more than a control condition of self-generated movements. We therefore suggest that the primary motor area is subdivided on the basis of anatomy, neurochemistry and function.

Inorganic, monovalent cations compete with agonists for the transmitter binding site of nicotinic acetylcholine receptors

The properties of adult mouse recombinant nicotinic acetylcholine receptors activated by acetylcholine (ACh+) or tetramethylammonium (TMA+) were examined at the single-channel level. The midpoint of the dose-response curve depended on the type of monovalent cation present in the extracellular solution. The shifts in the midpoint were apparent with both inward and outward currents, suggesting that the salient interaction is with the extracellular domain of the receptor. Kinetic modeling was used to estimate the rate constants for agonist binding and channel gating in both wild-type and mutant receptors exposed to Na+, K+, or Cs+. The results indicate that in adult receptors, the two binding sites have the same equilibrium dissociation constant for agonists. The agonist association rate constant was influenced by the ionic composition of the extracellular solution whereas the rate constants for agonist dissociation, channel opening, and channel closing were not. In low-ionic-strength solutions the apparent association rate constant increased in a manner that suggests that inorganic cations are competitive inhibitors of ACh+ binding. There was no evidence of an electrostatic potential at the transmitter binding site. The equilibrium dissociation constants for inorganic ions (Na+, 151 mM; K+, 92 mM; Cs+, 38 mM) and agonists (TMA+, 0.5 mM) indicate that the transmitter binding site is hydrophobic. Under physiological conditions, about half of the binding sites in resting receptors are occupied by Na+.

Quantitative receptor autoradiography of eight different transmitter-binding sites in the hippocampus of the common marmoset, Callithrix jacchus.

The regional and laminar distributions of eight different transmitter-binding sites were measured in the marmoset hippocampus by means of quantitative in vitro receptor autoradiography. Receptors for 5-HT1, L-glutamate, N-methyl-D-aspartate (NMDA) and GABAA were similarly distributed. The highest concentrations of these receptors were found in the pyramidal layer of CA1 and the molecular layer of the dentate gyrus. The 5-HT2 receptors showed the highest concentrations in the oriens layer of CA2. The highest concentrations of muscarinic M1 receptors were seen in the pyramidal layer of CA1. Muscarinic M2 receptors were most densely concentrated in the pyramidal layers of CA1, CA2 and CA3. The noradrenergic alpha 1 receptors were most densely packed in the radiatum-lacunosum-molecular layer of CA2 and the molecular layer of the dentate gyrus. Statistically significant co-distributions of serotoninergic, glutamatergic and GABAergic receptors point to possible interactions between these receptor systems in the same hippocampal regions and layers. Comparisons of marmoset distribution patterns for GABAA, NMDA, L-glutamate and 5-HT1 receptors with those in human hippocampi and those of other primates showed similarities between them. Clear differences in the patterns of alpha 1, M1, M2 and 5-HT2 receptors could be seen between marmoset and human hippocampi, indicating a high degree of species specificity in a presumably "conservative" brain region. More similarities, however, could be found between marmoset and human hippocampi than between those of rat and human brains, especially in relation to 5-HT1 and GABAA receptors and L-glutamate-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Desensitization is a property of the cholinergic binding region of the nicotinic acetylcholine receptor, not of the receptor-integral ion channel

The reversible acetylcholine esterase inhibitor (−)-physostigmine (eserine) is the prototype of a new class of nicotinic acetylcholine receptor (nAChR) activating ligands: it induces cation fluxes into nAChR-rich membrane vesicles from Torpedo marmorala electric tissue even under conditions of antagonist blocked acetylcholine binding sites (Okonjo, Kuhlmann, Maclicke, Neuron, in press). This suggests that eserine exerts its channel-activating property via binding sites at the nAChR separate from those of the natural transmitter. We now report that eserine can activate the channel even when the receptor has been preincubated (desensitized) with elevated concentrations of acetylcholine. Thus the conformational state of the receptor corresponding to desensitization is confined to the transmitter binding region, leaving the channel fully activatable — albeit only from other than the transmitter binding site(s).

Quantitative auto radiography of 11 different transmitter binding sites in the basal forebrain region of the rat—Evidence of heterogeneity in distribution patterns

The distribution of 12 different binding sites for acetylcholine, l-glutamate, GABA, 5-hydroxytryptamine, dopamine and noradrenaline was measured with quantitative receptor autoradiography in four regions of the rat basal forebrain (medial septal nucleus including vertical and horizontal limbs of the diagonal band of Broca, magnocellular preoptic nucleus, substantia innominata and basal nucleus of Meynert, ventral pallidum). l-Glutamate binding sites represent the largest portion of the analysed receptors in all regions, followed by muscarinic 2, 5-hydroxytryptamine, and GABA A receptors. Muscarinic 1, dopamine 1, dopamine 2 and 5-hydroxytryptamine 2 receptors and α 1-, α 1A- and α 1B-adrenoceptors represent the minor receptor populations. The largest portion of the dopamine receptors is represented by the dopamine, subtype, and the α 1B subtype dominates the α 1-adrenoceptor group. A heterogeneity of the distribution patterns of the different receptors throughout the basal forebrain regions is found. A comparison of the patterns shows that α 1-adrenoceptors have a similar regional distribution to that of the muscarinic, receptors, but both receptor types have reciprocal distributions compared with the 5-hydroxytryptamine 1 receptors. The results indicate that one transmitter may exert different effects in the basal forebrain regions depending on the densities of the respective receptor subtypes. Moreover, similar or reciprocal distribution patterns of some, but not all, analysed receptors point to a non-random association (co-distribution) of the different transmitter systems in the basal forebrain regions.

The expression of excitatory amino acid binding sites during neuritogenesis in the developing rat cerebellum

The present study has examined excitatory amino acid transmitter binding sites as measured autoradiographically in cryostat sections prepared from developing rat cerebella during the period of granule cell neuritogenesis. The external germinal layer (EGL) and molecular layer (ML), which during development contain granule cells at early stages of axon growth, contained only low levels of NMDA-displaceable l-[ 3H]glutamate binding sites. Similarly, [ 3H]glycine binding to the NMDA receptor linked binding site was not enriched in the EGL. Radioligand binding to the NMDA receptor was always greater in the granular layer (GL) than in the ML. The developmental increases in NMDA-displaceable l-[ 3H]glutamate and in [ 3H]glycine binding to the GL were similar but NMDA displaceable l-[ 3H]glutamate binding density increased before [ 3H]glycine binding sites. Glycine increased NMDA-displaceable l-[ 3H]glutamate binding only in the adult cerebellum. These results suggest that NMDA stimulation of neuritogenesis in granule cell cultures may reflect stimulation of dendritogenesis in the developing glomerulus rather than a stimulation of axon growth in the EGL. Also, NMDA receptors may be present in an immature form during cerebellar development and have different properties to the adult receptor. Binding sites for [ 3H]kainate and [ 3H]AMPA were present in both the GL and ML and increased during development. At all times the amount of binding sites for [ 3H]kainate were highest in the GL whereas those for [ 3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionate were highest in the ML.

Neurotransmitter receptors of the rolling mouse Nagoya: a quantitative autoradiographic study

The distribution of neurotransmitter and neuromodulator receptors was studied in the brain of the rolling mouse Nagoya (RMN) and in controls, using in vitro receptor autoradiography. Quantitative autoradiography was used to map adenosine A 1 (labeled with [ 3H]cyclohexyladenosine), GABA A ([ 3H]muscimol), opiate ([ 3H]naloxone), l-glutamate ([ 3H] l-glutamate), benzodiazepine ([ 3H]flunitrazepam), and muscarinic cholinergic ([ 3H]quinuclidinyl benzilate) receptors. In the cerebellar cortex, GABA A and adenosine A 1 binding sites were significantly reduced in the RMN, whereas other transmitter binding sites were not significantly altered. Adenosine A 1 binding sites were also reduced in the cerebral cortex and caudate-putamen. Benzodiazepine binding was significantly decreased in the cerebral cortex and increased in the CA1 subfield of the hippocampus. These results suggest that neurochemical alterations in the caudate-putamen as well as in the cerebellar cortex play important roles in the ataxia and motor dysfunction of the RMN.

Quantitative autoradiography of transmitter binding sites with an image analyzer

A computerized image processing system for quantitative receptor autoradiography on tritium-sensitive film is described. The method implements an image analyzer ( IBAS 1 + 2) for the acquisition of an image which is evaluated with respect to the regional distribution of binding site densities by a small computer which is connected to a digitizer. Several methodological problems must be considered when using tritium-labelled compounds and an image analyzer. The use of tissue standards and shading procedures, the improvement of signal-to-noise ratio, the calibration of the system which establishes the non-linear relation between grey values and concentration of radioactivity, the contrast enhancement and the quenching problem are discussed. This method permits a quick and precise quantitative analysis of binding site distribution in receptor autoradiography.

Solubilization and Characterization of Histamine H1 receptors in brain.

[3H]Doxepin, a tricyclic antidepressant, binds with high affinity to guinea pig brain membranes with a drug specificity indicating an association with H1 histamine receptors. The [3H]doxepin binding site has been solubilized, with digitonin being the only detergent able to maintain specific binding after solubilization. After solubilization, the kinetics and drug specificity of binding are virtually identical with those obtained in the intact membranes, indicating a conservation of the transmitter binding site after removal of the receptor from its lipid environment. The regulation of agonist affinity for the histamine H1 receptor by cations is maintained after solubilization. Sodium and to a similar extent lithium, but not potassium, rubidium, or cesium, decrease the affinity of agonists for the receptor. The divalent cations manganese and magnesium maintain their ability to increase agonist affinity after solubilization. Guanine nucleotides, however, lose their ability to decrease agonist affinity for the histamine H1 receptor after solubilization. Histamine receptors in rat brain differ from guinea pig brain receptors in the potency of several antihistamines. The difference is maintained in the solubilized receptors. Sucrose gradient and gel filtration experiments indicated Mr approximately 430,000 for the receptor-digitonin complex. The isoelectric point of the receptor is 4.8. None of these physical techniques distinguishes between guinea pig and rat brain receptors.

Identification of synapse specific components: synaptic glycoproteins, proteins, and transmitter binding sites.

Synaptic junctions (SJ) were prepared from synaptic plasma membranes (SPM) by extraction with Triton X-100 and density gradient centrifugation. These SJs were enriched in certain Concanavalin A (Con A) binding glycoproteins, the 52,000 Mr postsynaptic density (PSD) protein, and receptor sites for L-glutamate, L-aspartate, kainic acid (KA) but not quinuclidinyl benzilate (QNB). Various other membrane fractions were extracted by means of the same procedure. Those fractions prepared from light SPMs and crude myelin contained identifiable synaptic junctions and were also highly enriched in the synaptic components. The SJ-like fraction from mitochondria did not contain any of the characteristic synaptic macromolecules. However, this fraction from microsomes contained levels of the 52,000 Mr PSD protein and binding sites for L-glutamate (L-Glu) and L-aspartate (L-Asp) similar to true synaptic junctions, although the Con A binding glycoproteins and KA binding sites were nearly absent. On the basis of electron microscopy, the SJ-like fraction from microsomes did not contain structures recognizable as SJs. Thus, the Con A binding glycoproteins and KA binding sites appear to be excellent markers for the SJ.

Reconstitution of active acetylcholine receptor by hybridisation of binding site-blocked with ion channel-blocked acetylcholine receptor protein

The nicotinic acetylcholine receptor regulates the ion permeability of the postsynaptic membrane. This report presents evidence that the transmitter binding site and the ion channel may be located on distinct subunits. By hybridisation of receptor complexes, in which the transmitter binding site was blocked with complexes in which the ion channel was irreversibly inhibited, we reconstituted active acetylcholine receptor complexes. The reconstituted system was similar to the native receptor in its ability to regulate the ion permeability of lipid vesicles in response to nicotinic cholinergic effectors.