The conductance of the muscle nicotinic receptor channel changes rapidly upon gating.

We have recorded single-channel currents through fetal-type muscle nicotinic receptor channels at recording bandwidths of approximately 50 and 75 kHz. The time course of the rising phase of aligned and averaged openings can be entirely accounted for if it is assumed that the conductance of the single channel changes instantaneously, and that alignment and averaging introduce a dispersion of 2-3 microseconds. We conclude that we find no evidence for a gradual change in conductance as a channel opens or closes. The shapes of averaged power spectra are consistent with this conclusion, insofar as they exclude an exponential relaxation in the transition with a time constant of 10 microseconds or more.     

Photoactivation and dissociation of agonist molecules at the nicotinic acetylcholine receptor in voltage-clamped rat myoballs.

The photochemical properties of the azobenzene derivative, Bis-Q, were exploited to carry out an agonist concentration jump followed by a molecular rearrangement of bound agonist molecules at acetylcholine (ACh) receptor channels of voltage-clamped rat myoballs. Myoballs were bathed in solutions containing low concentrations of cis-Bis-Q, the inactive isomer. Whole-cell current relaxations were studied following a light flash that produced a concentration jump of agonist, trans-Bis-Q, followed by a second flash that produced net trans----cis photoisomerizations of Bis-Q molecules. The concentration-jump relaxation provided a measure of the mean burst duration for ACh receptor channels occupied by trans-Bis-Q (7.7 ms, 22 degrees C). The second current relaxation was a more rapid conductance decrease (phase 1, tau = 0.8 ms). Phase 1 may represent either the burst duration for receptors initially occupied by a single cis- and a single trans-Bis-Q molecule or that for unliganded receptors. Single-channel current recordings from excised outside-out membrane patches showed that single channels open following an agonist concentration jump comparable to that used in the whole-cell experiments; when many such records were averaged, a synthetic macroscopic relaxation was produced. Individual open channels closed faster following a flash that promoted trans----cis photoisomerizations of the bound ligand, thus confirming the whole-cell observations of phase 1.     

Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an alpha(2)betaepsilondelta composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15' of the M1 domain is phenylalanine in alpha subunits while it is isoleucine in non-alpha subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant alpha subunit (alphaF15'I) as well as those containing the reverse mutations in the non-alpha subunits (betaI15'F, deltaI15'F, and epsilonI15'F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the beta subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of betaI15'F of AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant in the diliganded receptor, and a significant increase opening in the absence of agonist. Mutations at betaI15' showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an approximately 70% closed-state-like environment for the beta15' position at the transition state of gating. The overall results identify position 15' as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.     

The selectivity filter of a ligand-gated ion channel. The helix-M2 model of the ion channel of the nicotinic acetylcholine receptor

Evidence from electrophysiology and biochemistry supports the hypothesis that the ion channel of the nicotinic acetylcholine receptor is formed by homologous amino acid sequences of all receptor subunits, called helices M2. A model of the ion channel is proposed and the selectivity filter is described as a ring of negatively-charged amino acid side chains [(1988) Nature 335, 645-648] which may undergo conformational changes upon permeation of the cation.     

The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity.

Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.     

Multidimensional on-line screening for ligands to the alpha3beta4 neuronal nicotinic acetylcholine receptor using an immobilized nicotinic receptor liquid chromatographic stationary phase

The alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) subtype was immobilized on a liquid chromatographic support and the resulting column used for the rapid and direct on-line screening for nAChR ligands. A multidimensional chromatographic system was developed consisting of the immobilized receptor column (NR column) connected via a switching valve to a C(18) column that was, in turn, connected to a single quadrupole mass spectrometer. A mixture of 18 compounds, containing alpha3beta4 nAChR (7) and compounds that are not alpha3beta4 nAChR ligands (11), was injected onto the NR column. The mobile phase consisted of ammonium acetate (10 mM, pH 7.4)-methanol (95:5, v/v) and the flow-rate was 0.2 ml/min. For the first 8 min the eluent was directed to waste. At t=8 min, the switching valve was rotated and the NR column connected to the C(18) column. The eluent from the NR column was directed to the C(18) column for 12 min. At t=20 min, the switching valve was rotated and the NR column was disconnected from the C(18) column. The compounds trapped on the C(18) column were separated and eluted onto the mass spectrometer using a mobile phase of ammonium acetate (10 mM, pH 7.4)-methanol (40:60, v/v) at a flow-rate of 1.0 ml/min. Detection was accomplished using total ion monitoring. The multidimensional system correctly isolated six of the seven alpha3beta4 nAChR ligands and only one of the 11 non-ligands was found with the alpha3beta4 nAChR ligands. The results indicate that the multidimensional liquid chromatographic system can be used for the on-line screening of chemical mixtures for alpha3beta4 nAChR ligands.     

The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical beta subunits

The saxitoxin receptor of the sodium channel purified from rat bran contains three types of subunits: alpha with Mr approximately 270,000, beta 1 with Mr approximately 39,000, and beta 2 with Mr approximately 37,000. These are the only polypeptides which quantitatively co-migrate with the purified saxitoxin receptor during velocity sedimentation through sucrose gradients. beta 1 and beta 2 are often poorly resolved by gel electrophoresis in sodium dodecyl sulfate (SDS), but analysis of the effect of beta-mercaptoethanol on the migration is covalently attached to the alpha subunit by disulfide bonds while the beta 1 subunit is not. The alpha and beta subunits of the sodium channel were covalently labeled in situ in synaptosomes using a photoreactive derivative of scorpion toxin. Treatment of SDS-solubilized synaptosomes with beta-mercaptoethanol decreases the apparent molecular weight of the alpha subunit band without change in the amount of 125I-labeled scorpion toxin associated with either the alpha or beta subunit bands. These results indicate that the alpha and beta 1 subunits are labeled by scorpion toxin whereas beta 1 is not and that the beta 2 subunit is covalently attached to alpha by disulfide bonds in situ as well as in purified preparations.     

The role of charge in lipid selectivity for the nicotinic acetylcholine receptor.

The effect of salt and pH titration on the selectivity of spin-labeled analogues of phosphatidic acid, phosphatidylserine, phosphatidylcholine, and stearic acid for the nicotinic acetylcholine receptor (nAcChoR) reconstituted into dioleoylphosphatidylcholine was examined at 0 degrees C using electron spin resonance spectroscopy. The order of selectivity at pH 7.4 and 0 mM NaCl was phosphatidylserine > stearic acid > phosphatidic acid > phosphatidylcholine. The addition up to 2 M NaCl or titration of pH from 5.0 to > 9.0 did not alter the selectivity of the phospholipids for the nAcChoR. For stearic acid, conversely, titration of pH from 5.0 to 9.0 at 0 mM NaCl and titration of NaCl from 0 to 2 M at pH 9.0 both increased selectivity for the nAcChoR. It is concluded that electrostatic interactions do not account for the selectivity of the negatively charged phospholipids, phosphatidylserine, and phosphatidic acid for the nAcChoR. This is consistent with the known orientation of the transmembrane sequences M1 and M4, which predicts a balance in the number of negative and positive charges in the lipid-protein interface and suggests that the two positive charges on each M3 helix are not exposed to the lipid-protein interface.     

Cyclodiene resistance at the insect GABA receptor/chloride channel complex confers broad cross resistance to convulsants and experimental phenylpyrazole insecticides

This study investigated the pharmacological profile of cyclodiene resistance in Drosophila melanogaster and the mode of action of a phenylpyrazole insecticide, JKU 0422. Toxicological studies were performed with a sucrose bait assay containing the synergist piperonyl butoxide. The Maryland strain of D. melanogaster was resistant to dieldrin, lindane, picrotoxinin, TBPS, p-CN-TBOB, and JKU 0422. In contrast, this strain was susceptible to cypermethrin and the avermectins MK-243, abamectin, and abamectin 8,9-oxide. Neurophysiological studies showed that both TBPS and JKU 0422 reversed the inhibitory action of GABA in central nerve preparations from susceptible D. melanogaster. However, the response to these compounds was attenuated in nerve preparations from the resistant Maryland strain, which indicated that the resistance was expressed at the level of the nerve. Topical toxicity bioassays with JKU 0422 on susceptible (CSMA) and cyclodiene-resistant (LPP) strains of German cockroach revealed a resistance ratio of 553-fold for this compound. These studies demonstrate that cyclodiene resistance in D. melanogaster confers broad cross resistance toward compounds thought to block the GABA-gated chloride channel in a manner similar to the cyclodienes. Moreover, the cross resistance extends to JKU 0422, and resistance to this compound is also present in a strain of cyclodiene-resistant German cockroach. These toxicological results, along with the neurophysiological studies, confirm that JKU 0422 has a mode of action that is similar to the cyclodienes and TBPS. These findings suggest that the introduction and use of new chloride channel antagonists as insecticides should be managed carefully in order to prevent the rapid development of resistance in the field. © 1994 Wiley-Liss, Inc.     

The organized chromatin domain of the repressed yeast a cell-specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome

In yeast alpha cells the a cell-specific genes STE6 and BAR1 are packaged as gene-sized chromatin domains of positioned nucleosomes. Organized chromatin depends on Tup1p, a corepressor that interacts with the N-terminal regions of H3 and H4. If Tup1p functions to organize or stabilize a chromatin domain, the protein might be expected to be present at a level stoichiometric with nucleosomes. Chromatin immunoprecipitation assays using Tup1p antibodies showed Tup1p to be associated with the entire genomic STE6 coding region. To determine stoichiometry of Tup1p associated with the gene, a yeast plasmid containing varying lengths of the STE6 gene including flanking control regions and an Escherichia coli lac operator sequence was constructed. After assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromosomes were isolated using an immobilized lac repressor. In these experiments, Tup1p was found to be specifically associated with repressed STE6 chromatin in vivo at a ratio of about two molecules of the corepressor per nucleosome. These observations strongly suggest a structural role for Tup1p in repression and constrain models for organized chromatin in repressive domains.     

Functional dissection of structural domains in the receptor for colony-stimulating factor-1.

Receptor tyrosine kinases (RTKs) transduce external signals to the interior of the cell via a cytoplasmic kinase domain. We demonstrated previously that ligand-induced kinase activation of the colony-stimulating factor-1 receptor (CSF-1R) occurs via receptor oligomerization without propagation of conformational changes through the transmembrane (TM) domain (Lee, A. W., and Nienhuis, A. W. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7270-7274). We have now examined the role of the different subdomains in the metabolic and signaling properties of CSF-1R. Two types of chimeric receptors have been utilized, Glyfms A, with the extracellular and TM domains of glycophorin A (GpA) and the cytoplasmic domain of CSF-1R, and Glyfms B, where only the extracellular domain originates from GpA. Glyfms A was found to exhibit a higher basal level of in vitro kinase activity, an increased associated phosphatidylinositol (PtdIns) 3-kinase activity and to support enhanced cellular mitogenesis, compared with wild-type CSF-1R or to Glyfms B. The constitutive activation of Glyfms A is consistent with the hypothesis that the TM domain may play a role in receptor oligomerization. Cross-linking with anti-GpA antibodies activated the kinase function of Glyfms B leading to an increase in PtdIns 3-kinase association and to the transmission of a mitogenic signal. Our results indicate that an activated kinase domain contains the major determinant for coupling with PtdIns 3-kinase, independent of extracellular and TM sequences and of ligand binding. Both chimeric receptors underwent internalization in the presence of anti-GpA antibodies but were not degraded, including the tyrosine-phosphorylated and kinase-active population. These results suggest that structural determinants in the extracellular domain must be important for targeting internalized receptors for lysosomal degradation.     

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells.

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-1 antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. There is no apparent relationship between amounts of EBNA-1 and viral DNA.     

Equilibrium ligand binding to the human erythrocyte sugar transporter. Evidence for two sugar-binding sites per carrier

Equilibrium [3H]cytochalasin B binding to class I sites of human red cell membranes (the sugar transporter) was examined in the presence and absence of intracellular or extracellular sugars known to interact with the transport system. D-Glucose, a transported sugar, is without effect on cytochalasin B binding when present in the extracellular medium but is an effective inhibitor of binding when present within the cell. Ethylidene glucose and maltose (reactive but nontransported sugars) inhibit cytochalasin B (CCB) binding when present either outside or inside the red cell. Inhibition by intracellular sugar (Si) is of the simple, linear competitive type. Inhibition by extracellular sugars (So) is more complex; the Kd(app) for cytochalasin B binding increases in a saturable fashion with [So]. These observations are compared with the predictions of the one-site, alternating conformer model and the two-site model for substrate binding to the sugar transporter, X. The experimental results are inconsistent with the one-site model but are explained by a two-site model in which the ternary complexes of So . X . Si or So . X . CCBi exist and where the binding sites for So and Si display negative cooperativity when occupied by nontransported substrate and little or no cooperativity when occupied by the transported species, D-glucose.     

A partial structure for the gamma-aminobutyric acid (GABAA) receptor is derived from the model for the nicotinic acetylcholine receptor. The anion-exchange protein of cell membranes is related to the GABAA receptor

Based on the nicotinic acetylcholine receptor model [(1987) Eur. J. Biochem. 168, 431-449], a partial model is constructed for the exobilayer portion of the GABAA receptor, an approach justified by the superfamily relationship of the two receptors [(1987) Nature 328, 221-227]. The model predicts successfully the excess positive charge on interior strands which constitute the ligand-responsive portion of the receptor. Binding to GABA expands the exobilayer portion of the receptor, opening a pathway to a chloride channel. Separate binding sites for antianxiolytics (benzodiazepines) and hypnotics (barbiturates) are suggested, with prolongation of chloride entry projected as a consequence of stabilization of the open form. The anion-exchange protein (AEP) of membranes (band 3 of red blood cell membranes) is similar in some respects to the gamma-aminobutyric acid (GABAA) receptor. Both proteins are inhibited and labeled by diisocyanatostilbenedisulfonate (DIDS), both transport Cl- and HCO-3, and both are membrane proteins. Starting with the lysines known to be labeled in band 3 protein, searches of the amino acid sequences of the GABAA receptor alpha- and beta-subunits reveal at least 4 reasonably homologous sequences. The relationship between AEP and GABAA receptor leads to the idea that the chloride/bicarbonate channel may be the ancestor of all ligand-gated channels, with ligand gating by gamma-aminobutyric acid and acetylcholine arising later in evolution.     

Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants,in pulmonary epithelial cells

The cation channel TRPA1 functions as a chemosensory protein and is directly activated by a number of noxious inhalants. A pulmonary expression of TRPA1 has been described in sensory nerve endings and its stimulation leads to the acceleration of inflammatory responses in the lung. Whereas the function of TRPA1 in neuronal cells is well defined, only few reports exist suggesting a role in epithelial cells. The aim of the present study was therefore (1) to evaluate the expression of TRPA1 in pulmonary epithelial cell lines, (2) to characterize TRPA1-promoted signaling in these cells, and (3) to study the extra-neuronal expression of this channel in lung tissue sections. Our results revealed that the widely used alveolar type II cell line A549 expresses TRPA1 at the mRNA and protein level. Furthermore, stimulating A549 cells with known TRPA1 activators (i.e., allyl isothiocyanate) led to an increase in intracellular calcium levels, which was sensitive to the TRPA1 blocker ruthenium red. Investigating TRPA1 coupled downstream signaling cascades it was found that TRPA1 activation elicited a stimulation of ERK1/2 whereas other MAP kinases were not affected. Finally, using epithelial as well as neuronal markers in immunohistochemical approaches, a non-neuronal TRPA1 protein expression was detected in distal parts of the porcine lung epithelium, which was also found examining human lung sections. TRPA1-positive staining co-localized with both epithelial and neuronal markers underlining the observed epithelial expression pattern. Our findings of a functional expression of TRPA1 in pulmonary epithelial cells provide causal evidence for a non-neuronal TRPA1-mediated control of inflammatory responses elicited upon TRPA1-mediated registration of toxic inhalants in vivo.     

The effects of vanilloid analogues structurally related to capsaicin on the transient receptor potential vanilloid 1 channel

Transient receptor potential vanilloid 1 (TRPV1) is known as a receptor of capsaicin, a spicy ingredient of chili peppers. It is also sensitive to a variety of pungent compounds and is involved in nociception. Here, we focused on the structural characteristics of capsaicin, and investigated whether vanillylmanderic acid (VMA), vanillic acid (VAcid), vanillyl alcohol (VAlc), vanillyl butyl ether (VBE), and vanillin, containing a vanillyl skeleton similar to capsaicin, affected the TRPV1 activities. For detection of TRPV1 activity, intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in HEK 293 cells heterologously expressing mouse TRPV1 (mTRPV1-HEK) and in mouse sensory neurons. Except for vanillin, four vanilloid analogues dose-dependently increased [Ca²⁺]_i in mTRPV1-HEK. The solutions that dissolved VMA, VAcid and vanillin at high concentrations were acidic, whereas those of VAlc and VBE were neutral. Neutralized VAcid evoked [Ca²⁺]_i increases but neutralized VMA did not. Mutation of capsaicin-sensing sites diminished [Ca²⁺]_i responses to VAcid, VAlc and VBE. VAcid, VMA, and vanillin suppressed the activation of TRPV1 induced by capsaicin. VAcid and VMA also inhibited the acid-induced TRPV1 activation. In sensory neurons, VMA diminished TRPV1 activation by capsaicin or acids. The present data indicate that these structural characteristics of chemical compounds on TRPV1 may provide strategies for the development of novel analgesic drugs targeting nociceptive TRPV1.