Selective modulation of neuronal nicotinic acetylcholine receptor channel subunits by G(o)-protein subunits

protein modulation of neuronal nicotinic acetylcholine receptor ( nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTP gamma S increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTP gamma S. In inside-out membrane patches, the open probability (NPo) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2mM GTP gamma S relative to control but was unchanged in the presence of GDP gamma S. The modulation of nAChR-mediated whole- cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTP gamma S. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or G beta gamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nM G beta gamma increased the open probability of ACh- activated single-channel currents fivefold, whereas G(o)alpha( 50 nM) produced no significant increase in NPo. Neuronal nAChR subunits alpha 3-alpha 5 and alpha 2 exhibited a positive interaction with G(o)alpha and G beta gamma, whereas beta 4 and alpha 7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons

1 The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca2+ concentration ([Ca2+](i)) and membrane currents were investigated in neonatal rat intracardiac neurons. 2 In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca2+](i), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca2+](i) transients was 28 muM, close to the estimated clinical EC50 (clinically relevant (half-maximal) effective concentration) of thiopental. 3 In fura-2-loaded neurons, voltage clamped at -60mV to eliminate any contribution of voltage-gated Ca2+ channels, thiopental (25 muM) simultaneously inhibited nAChR-induced increases in [Ca2+](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by - 40% at - 120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. 4 The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC50 were also shown to inhibit nAChR-induced increases in [Ca2+](i) by similar to40%. 5 Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca2+](i), indicating that inhibition of Ca2+ release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. 6 Depolarization-activated Ca2+ channel currents were unaffected in the presence of thiopental (25 muM), pentobarbital (50 muM) and ketamine (10 muM). 7 In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca2+](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.

The effects of alanine-substituted conantokin-G and ifenprodil on the human spermine-activated N-methyl-D-aspartate receptor

We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [H-3]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degreesC until assay. Both Con-GA7 and ifenprodil inhibited [H-3]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC50 changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC50 with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [H-3]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC50, while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-D-aspartate receptor. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

Contribution of the collagen I alpha 1 and vitamin D receptor genes to the risk of hip fracture in elderly women

Context and Objective: Hip fracture is partially genetically determined. The present study was designed to examine the contributions of vitamin D receptor (VDR) and collagen I alpha 1 (COLIA1) genotypes to the liability to hip fracture in postmenopausal women. Design: The study was designed as a prospective population-based cohort investigation. Subjects: Six hundred seventy-seven postmenopausal women of Caucasian background, aged 70 +/- 7 yr (mean +/- SD), have been followed for up to 14 yr. Sixty-nine women had sustained a hip fracture during the period. Main Outcome: Atraumatic hip fractures were prospectively identified through radiologists' reports. Bone mineral density (BMD) at the hip and lumbar spine was measured by dual-energy x-ray absorptiometry. Genotypes: The TaqI and SpI COLIA1 polymorphisms of the VDR and COLIA1 genes were determined. Using the Single Nucleotide Polymorphism database, VDR TT, Tt, and tt genotypes were coded as TT, TC, and CC, whereas COLIA1 SS, Ss, and ss were coded as GG, GT, and TT. Results: Women with VDR CC genotype (16% prevalence) and COLIA1 TT genotype (5% prevalence) had an increased risk of hip fracture [odds ratio (OR) associated with CC, 2.6; 95% confidence interval (CI), 1.2-5.3; OR associated with TT, 3.8; 95% CI, 1.3-10.8] after adjustment for femoral neck BMD (OR, 3.4 per SD; 95% CI, 2.3-5.0) and age (OR, 1.4 per 5 yr; 95% CI, 1.1-1.7). Approximately 20 and 12% of the liability to hip fracture was attributable to the presence of the CC genotype and TT genotype, respectively. Conclusion: The VDR CC genotype and COLIA1 TT genotype were associated with increased hip fracture risk in Caucasian women, and this association was independent of BMD and age.

Papillomavirus virus-like particles activate the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding

We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 3 1, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3 beta. We also present evidence that PV signaling is activated via the alpha 6 beta 4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection. (c) 2006 Elsevier Inc. All rights reserved

Alpha-selenoconotoxins, a new class of potent alpha(7) neuronal nicotinic receptor antagonists

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and non-reducible diselenide bonds. Three isoforms of alpha-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one([ Sec(2,8)] ImI or [Sec(3,12)] ImI), or both([Sec(2,3,8,12)] ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native ImI, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the alpha(7) nicotinic acetylcholine receptor, with each seleno-analogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs.