Spontaneous activity of neostriatal cholinergic interneurons in vitro.
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Neostriatal cholinergic interneurons fire irregularly but tonically in vivo. The summation of relatively few depolarizing potentials and their temporal sequence are thought to underlie spike triggering and the irregularity of action potential timing, respectively. In these experiments we used whole-cell, cell-attached, and extracellular recording techniques to investigate the role of spontaneous synaptic inputs in the generation and patterning of action potentials in cholinergic interneurons in vitro. Cholinergic cells were spontaneously active in vitro at 25 +/- 1 degrees C during whole-cell recording from 2 to 3 week postnatal slices and at 35 +/- 2 degrees C during cell-attached and extracellular recording from 3 to 4 week postnatal slices. A range of firing frequencies and patterns was observed including regular, irregular, and burst firing. Blockade of AMPA and NMDA receptors altered neither the firing rate nor the pattern, and accordingly, voltage-clamp data revealed a very low incidence of spontaneous EPSCs. GABAA receptor antagonists were also ineffective in altering the spiking frequency or pattern owing to minimal inhibitory input in vitro. Functional excitatory and inhibitory inputs to cholinergic cells were disclosed after application of 4-aminopyridine (100 microM), indicating that these synapses are present but not active in vitro. Blockade of D1 or D2 dopamine receptors or muscarinic receptors also failed to influence tonic activity in cholinergic cells. Together these data indicate that cholinergic interneurons are endogenously active and generate action potentials in the absence of any synaptic input. Interspike interval histograms and autocorrelograms generated from unit recordings of cholinergic cells in vitro were indistinguishable from those of tonically active neurons recorded in vivo. Irregular spiking is therefore embedded in the mechanism responsible for endogenous activity. (+info)
Signaling domain of the aspartate receptor is a helical hairpin with a localized kinase docking surface: cysteine and disulfide scanning studies.
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Cysteine and disulfide scanning has been employed to probe the signaling domain, a highly conserved motif found in the cytoplasmic region of the aspartate receptor of bacterial chemotaxis and related members of the taxis receptor family. Previous work has characterized the N-terminal section of the signaling domain [Bass, R. B., and Falke, J. J. (1998) J. Biol. Chem. 273, 25006-25014], while the present study focuses on the C-terminal section and the interactions between these two regions. Engineered cysteine residues are incorporated at positions Gly388 through Ile419 in the signaling domain, thereby generating a library of receptors each containing a single cysteine per receptor subunit. The solvent exposure of each cysteine is ascertained by chemical reactivity measurements, revealing a periodic pattern of buried hydrophobic and exposed polar residues characteristic of an amphipathic alpha-helix, denoted helix alpha8. The helix begins between positions R392 and Val401, then continues through the last residue scanned, Ile419. Activity assays carried out both in vivo and in vitro indicate that both the buried and exposed faces of this amphipathic helix are critical for proper receptor function and the buried surface is especially important for kinase downregulation. Patterns of disulfide bond formation suggest that helix alpha8, together with the immediately N-terminal helix alpha7, forms a helical hairpin that associates with a symmetric hairpin from the other subunit of the homodimer, generating an antiparallel four helix bundle containing helices alpha7, alpha7', alpha8, and alpha8'. Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices alpha7 and alpha8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking. (+info)
The aspartate receptor cytoplasmic domain: in situ chemical analysis of structure, mechanism and dynamics.
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BACKGROUND: Site-directed sulfhydryl chemistry and spectroscopy can be used to probe protein structure, mechanism and dynamics in situ. The aspartate receptor of bacterial chemotaxis is representative of a large family of prokaryotic and eukaryotic receptors that regulate histidine kinases in two-component signaling pathways, and has become one of the best characterized transmembrane receptors. We report here the use of cysteine and disulfide scanning to probe the helix-packing architecture of the cytoplasmic domain of the aspartate receptor. RESULTS: A series of designed cysteine pairs have been used to detect proximities between cytoplasmic helices in the full-length, membrane-bound receptor by measurement of disulfide-bond formation rates. Upon mild oxidation, 25 disulfide bonds from rapidly between three specific pairs of helices, whereas other helix pairs yield no detectable disulfide-bond formation. Further constraints on helix packing are provided by 14 disulfide bonds that retain receptor function in an in vitro kinase regulation assay. Of these functional disulfides, seven lock the receptor in the conformation that constitutively stimulates kinase activity ('lock-on'), whereas the remaining seven retain normal kinase regulation. Finally, disulfide-trapping experiments in the absence of bound kinase reveal large-amplitude relative motions of adjacent helices, including helix translations and rotations of up to 19 A and 180 degrees, respectively. CONCLUSIONS: The 25 rapidly formed and 14 functional disulfide bonds identify helix-helix contacts and their register in the full-length, membrane-bound receptor-kinase complex. The results reveal an extended, rather than compact, domain architecture in which the observed helix-helix interactions are best described by a four-helix bundle arrangement. A cluster of six lock-on disulfide bonds pinpoints a region of the four-helix bundle critical for kinase activation, whereas the signal-retaining disulfides indicate that signal-induced rearrangements of this region are small enough to be accommodated by disulfide-bond flexibility (< or = 1.2 A). In the absence of bound kinase, helix packing within the cytoplasmic domain is highly dynamic. (+info)
A piston model for transmembrane signaling of the aspartate receptor.
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To characterize the mechanism by which receptors propagate conformational changes across membranes, nitroxide spin labels were attached at strategic positions in the bacterial aspartate receptor. By collecting the electron paramagnetic resonance spectra of these labeled receptors in the presence and absence of the ligand aspartate, ligand binding was shown to generate an approximately 1 angstrom intrasubunit piston-type movement of one transmembrane helix downward relative to the other transmembrane helix. The receptor-associated phosphorylation cascade proteins CheA and CheW did not alter the ligand-induced movement. Because the piston movement is very small, the ability of receptors to produce large outcomes in response to stimuli is caused by the ability of the receptor-coupled enzymes to detect small changes in the conformation of the receptor. (+info)
High-affinity interaction of (des-Tyrosyl)dynorphin A(2-17) with NMDA receptors.
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The opioid peptide dynorphin A elicits non-opioid receptor-mediated, neurotoxic response in vivo, which is blocked by pretreatment with MK-801, a noncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist. In the present study, we examined the possible direct interaction of dynorphin A on the NMDAR. A nonopioid dynorphin A analog, (125)I-(des-tyrosyl) dynorphin A(2-17), was used in radioligand binding analysis on rat cortical brain membranes. This radioligand exhibited a saturable, specific binding at high affinity with a K(d) value of 9.4+/-1.6 nM and maximal binding of 2.4+/-0.6 pmol/mg protein. This binding site was associated with the NMDAR complex because it was modulated by a number of NMDAR ligands. Transient expression of the rat NR1a/NR2A complex in human embryonic kidney 293 cells confirmed a coexpression of (125)I-(des-tyrosyl) dynorphin A(2-17), [(3)H]CGP39,653, and [(3)H]MK-801 binding. These data provide direct evidence of the presence of a high-affinity binding site for dynorphin A on the NMDAR. The modulatory effect of the various NMDAR-selective ligands on dynorphin A binding suggests that dynorphin A may bind preferentially to the closed/desensitized state of the NMDAR. The physiological role of dynorphin A binding to the NMDAR remains to be established. (+info)
The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis.
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The Escherichia coli aspartate receptor is a dimer with two transmembrane sequences per monomer that connect a periplasmic ligand binding domain to a cytoplasmic signaling domain. The method of 'hydrophobic-biased' random mutagenesis, that we describe here, was used to construct mutant aspartate receptors in which either the entire transmembrane sequence or seven residues near the center of the transmembrane sequence were replaced with hydrophobic and polar random residues. Some of these receptors responded to aspartate in an in vivo chemotaxis assay, while others did not. The acceptable substitutions included hydrophobic to polar residues, small to larger residues, and large to smaller residues. However, one mutant receptor that had only a few hydrophobic substitutions did not respond to aspartate. These results add to our understanding of sequence specificity in the transmembrane regions of proteins with more than one transmembrane sequence. This work also demonstrates a method of constructing families of mutant proteins containing random residues with chosen characteristics. (+info)
Interactions between GABA and glycine at inhibitory amino acid receptors on rat olfactory bulb neurons.
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Whole cell voltage-clamp electrophysiology was used to examine interactions between GABA and glycine at inhibitory amino acid receptors on rat olfactory bulb neurons in primary culture. Membrane currents evoked by GABA and glycine were selectively inhibited by low concentrations of bicuculline and strychnine, respectively, suggesting that they activate pharmacologically distinct receptors. However, GABA- and glycine-mediated currents showed cross-inhibition when the two amino acids were applied sequentially. Application of one amino acid inhibited the response to immediate subsequent application of the other. In the majority of neurons, GABA inhibited subsequent glycine-evoked currents and glycine inhibited subsequent GABA-evoked currents. In a small proportion of neurons, however, GABA inhibited glycine-evoked currents but glycine had little effect on GABA-evoked currents. The reverse was true in other neurons, suggesting that alterations in chloride gradients alone did not account for the cross-inhibition. Furthermore, no cross-inhibition was observed between GABA- and glycine-evoked currents in some neurons. The amplitude of the current evoked by the coapplication of saturating concentrations of GABA and glycine in these neurons was nearly the sum of the currents evoked by GABA and glycine alone. In contrast, the currents were not additive in neurons demonstrating cross-inhibition. These results suggest that olfactory bulb neurons heterogeneously express a population of inhibitory amino acid receptors that can bind either GABA or glycine. Interactions between GABA and glycine at inhibitory amino acid receptors may provide a mechanism to modulate inhibitory synaptic transmission. (+info)
Attractant regulation of the aspartate receptor-kinase complex: limited cooperative interactions between receptors and effects of the receptor modification state.
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The manner by which the bacterial chemotaxis system responds to a wide range of attractant concentrations remains incompletely understood. In principle, positive cooperativity between chemotaxis receptors could explain the ability of bacteria to respond to extremely low attractant concentrations. By utilizing an in vitro receptor-coupled kinase assay, the attractant-dependent response curve has been measured for the Salmonella typhimurium aspartate chemoreceptor. The attractant chosen, alpha-methyl aspartate, was originally used to quantitate high receptor sensitivity at low attractant concentrations by Segall, Block, and Berg [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8987-8991]. The attractant response curve exhibits limited positive cooperativity, yielding a Hill coefficient of 1.7-2.4, and this Hill coefficient is relatively independent of both the receptor modification state and the mole ratio of CheA to receptor. These results disfavor models in which there are strong cooperative interactions between large numbers of receptor dimers in an extensive receptor array. Instead, the results are consistent with cooperative interactions between a small number of coupled receptor dimers. Because the in vitro receptor-coupled kinase assay utilizes higher than native receptor densities arising from overexpression, the observed positive cooperativity may overestimate that present in native receptor populations. Such positive cooperativity between dimers is fully compatible with the negative cooperativity previously observed between the two symmetric ligand binding sites within a single dimer. The attractant affinity of the aspartate receptor is found to depend on the modification state of its covalent adaptation sites. Increasing the the level of modification decreases the apparent attractant affinity at least 10-fold in the in vitro receptor-coupled kinase assay. This observation helps explain the ability of the chemotaxis pathway to respond to a broad range of attractant concentrations in vivo. (+info)