Chain length dependence of the interactions of bisquaternary ligands with the Torpedo nicotinic acetylcholine receptor.

The interactions of a series of bisholine esters [(CH3)3N+CH(2)CH2OCO-(CH2)n-COOCH2CH2N+(CH3)3] with the Torpedo nicotinic acetylcholine receptor have been investigated. In equilibrium binding studies, [3H]-suberyldicholine (n=6) binds to an equivalent number of sites as [3H]-acetylcholine and with similar affinity (KD approximately 15 nM). In competition studies, all bischoline esters examined displaced both radioligands in an apparently simple competitive manner. Estimated dissociation constants (KI) showed clear chain length dependence. Short chain molecules (n6) had high affinity similar to suberyldicholine. Functional responses were measured by either rapid flux techniques using Torpedo membrane vesicles or voltage-clamp analyses of recombinant receptors expressed in Xenopus oocytes. Both approaches revealed that suberyldicholine (EC50 approximately 3.4 microM) is 14-25-fold more potent than acetylcholine. However, suberyldicholine elicited only about 45% of the maximum response of the natural ligand, i.e., it is a partial agonist. The potency of this bischoline series increased with chain length. Whereas the shorter ligands (nor=4) had similar (or higher) potency to suberyldicholine. Ligand efficacy had an approximately bell-shaped dependence on chain length and compounds where nor=8 were very poor partial agonists. Based on estimates of interonium distances, we suggest that bisquaternary ligands can interact with multiple binding sites on the nAChR and, depending on the conformational state of the receptor, these sites are 15-20A apart.

Tryptophan 86 of the alpha subunit in the Torpedo nicotinic acetylcholine receptor is important for channel activation by the bisquaternary ligand suberyldicholine.

Suberyldicholine, a bisquaternary compound, is a potent nicotinic acetylcholine receptor agonist. Previously, we suggested that at least some of the unusual binding properties of this ligand may be a consequence of its ability to cross-link two binding "subsites" within each of the high-affinity agonist binding domains [Dunn, S. M. J., and Raftery, M. A. (1997) Biochemistry 36, 3846-3853]. Tryptophan 86 of the alpha subunit has previously been implicated in the binding of agonist to this receptor. However, on the basis of the crystal structure of a homologous acetylcholine binding protein, this residue is predicted to lie 15-20 A from the high-affinity site, i.e., a distance that approximates the interonium distance of suberyldicholine. Tryptophan 86 was mutated to either an alanine or a phenylalanine, and the mutated subunit was coexpressed with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Although the alanine mutation resulted in a loss of receptor expression, the alphaW86F mutant receptor was expressed on the oocyte surface, albeit with a much reduced efficiency. Acetylcholine-evoked currents of the alphaW86F receptor were not significantly different from those of the wild type with respect to the concentration dependence of channel activation, receptor desensitization, or d-tubocurarine inhibition. In contrast, the EC(50) for suberyldicholine-mediated activation of the alphaW86F receptor was increased by approximately 500-fold. Furthermore, suberyldicholine-evoked currents in the mutant receptor did not desensitize and were insensitive to block by d-tubocurarine. Thus, tryptophan 86 of the Torpedo receptor alpha subunit may be part of a subsite for recognition of suberyldicholine and other bisquaternary ligands.

Substantia Gelatinosa neurons in defined-medium organotypic slice culture are similar to those in acute slices from young adult rats.

Peripheral nerve injury promotes an enduring increase in the excitability of the spinal dorsal horn. This change, that likely underlies the development of chronic pain, may be a consequence of prolonged exposure of dorsal horn neurons to mediators such as neurotrophins, cytokines, and neurotransmitters. The long-term effects of such mediators can be analyzed by applying them to spinal neurons in organotypic slice culture. To assess the validity of this approach, we established serum-free, defined-medium organotypic cultures (DMOTC) from E13-14 prenatal rats. Whole-cell recordings were made from neurons maintained in DMOTC for up to 42 days. These were compared with recordings from neurons of similar age in acute spinal cord slices from 15- to 45-day-old rats. Five cell types were defined in acute slices as 'Tonic', 'Irregular', 'Delay', 'Transient' or 'Phasic' according to their discharge patterns in response to depolarizing current. Although fewer 'Phasic' cells were found in cultures, the proportions of 'Tonic', 'Irregular', 'Delay', and 'Transient' were similar to those found in acute slices. GABAergic, glycinergic, and 'mixed' inhibition were observed in neurons in acute slices and DMOTC. Pure glycinergic inhibition was absent in 7d cultures but became more pronounced as cultures aged. This parallels the development of glycinergic inhibition in vivo. These and other findings suggest that fundamental developmental processes related to neurotransmitter phenotype and neuronal firing properties are preserved in DMOTC. This validates their use in evaluating the cellular mechanisms that may contribute to the development of chronic pain.

Activation of the Torpedo nicotinic acetylcholine receptor. The contribution of residues alphaArg55 and gammaGlu93.

The Torpedo nicotinic acetylcholine receptor is a heteropentamer (alpha2betagammadelta) in which structurally homologous subunits assemble to form a central ion pore. Viewed from the synaptic cleft, the likely arrangement of these subunits is alpha-gamma-alpha-delta-beta lying in an anticlockwise orientation. High affinity binding sites for agonists and competitive antagonists have been localized to the alpha-gamma and alpha-delta subunit interfaces. We investigated the involvement of amino acids lying at an adjacent interface (gamma-alpha) in receptor properties. Recombinant Torpedo receptors, expressed in Xenopus oocytes, were used to investigate the consequences of mutating alphaArg55 and gammaGlu93, residues that are conserved in most species of the peripheral nicotinic receptors. Based on homology modeling, these residues are predicted to lie in close proximity to one another and it has been suggested that they may form a salt bridge in the receptor's three-dimensional structure (Sine et al. 2002 J Biol Chem277, 29 210-29 223). Although substitution of alphaR55 by phenylalanine or tryptophan resulted in approximately a six-fold increase in the EC50 value for acetylcholine activation, the charge reversal mutation (alphaR55E) had no significant effect. In contrast, the replacement of gammaE93 by an arginine conferred an eight-fold increase in the potency for acetylcholine-induced receptor activation. In the receptor carrying the double mutations, alphaR55E-gammaE93R or alphaR55F-gammaE93R, the potency for acetylcholine activation was partially restored to that of the wild-type. The results suggest that, although individually these residues influence receptor activation, direct interactions between them are unlikely to play a major role in the stabilization of different conformational states of the receptor.

Neurotrophic regulation of calcium channels by the peptide neurotransmitter luteinizing hormone releasing hormone.

We exploited the simple organization of bullfrog paravertebral sympathetic ganglia (BFSG) to test whether the neurotransmitter peptide luteinizing hormone releasing hormone (LHRH), which generates the late slow EPSP, could also exert long-term neurotrophic control of ion channel expression. Whole-cell recordings from B-cells in BFSG showed that removal of all of the sources of ganglionic LHRH for 10 d by cutting preganglionic C-fibers in vivo caused a 28% reduction in Ca2+ current density. When BFSG B-neurons were dissociated from adult bullfrogs and maintained in a defined-medium, neuron-enriched, low-density, serum-free culture, the ICa density was increased by 49% after 6-7 d in the presence of 0.45 microm LHRH. This increase was not associated with alterations in the voltage dependence of Ca2+ current activation or inactivation and reflected a selective increase in N-type Ca2+ channel current. The increase in ICa density induced by LHRH was blocked by the transcription inhibitor actinomycin D. These results suggest that chronic exposure to a neurotransmitter that acts through G-protein-coupled receptors exerts long-term control of ion channel expression in a fully differentiated, adult sympathetic neuron in vitro or in vivo.

Despite increased plasma concentration, inflammation reduces potency of calcium channel antagonists due to lower binding to the rat heart.

1. Rheumatoid arthritis reduces verapamil oral clearance thereby increases plasma concentration of the drug. This coincides with reduced drug effects through an unknown mechanism. 2. The effect of interferon-induced acute inflammation on the pharmacokinetics and electrocardiogram of verapamil (20 mg kg(-1), p.o.) and nifedipine (0.1 mg kg(-1), i.v.) was studied in Sprague-Dawley rats. 3. The effect of both acute and chronic inflammation on radioligand binding to cardiac L-type calcium channels was also investigated. 4. Acute inflammation resulted in increased plasma concentration of verapamil but had no effect on that of nifedipine. Verapamil binding to plasma proteins was unaffected. 5. As has been reported for humans, the increased verapamil concentration coincided with a reduction in the degree to which PR interval is prolonged by the drug. The effect of nifedipine on PR interval was also reduced by inflammation. 6. Maximum binding of (3)H-nitrendipine to cardiac cell membrane was significantly reduced from 63.2+/-2.5 fmol mg(-1) protein in controls to 46.4+/-2.0 in acute inflammation and from 66.8+/-2.2 fmol mg(-1) protein in controls to 42.2+/-2.0 in chronic inflammation. 7. Incubation of the normal cardiac cell membranes with 100 and 1000 pg ml(-1) of rat tissue necrosis factor-alpha did not influence the binding indices to the calcium channels. 8. Our data suggest that the reduced calcium channel responsiveness is because of altered binding to channels.