The inhibitory and facilitatory actions of amyloid-beta peptides on nicotinic ACh receptors and AMPA receptors

The present study investigated the effects of amyloid-beta peptides on nicotinic ACh receptors (Torpedo, alpha 4 beta 2, and alpha 7 receptors) and AMPA receptors expressed in Xenopus oocytes by monitoring whole-cell membrane currents. Ten-minutes treatment with amyloid-beta(1-42) (1 microM) inhibited Torpedo ACh receptor currents, reaching 53% of original levels 30 min after treatment. Amyloid-beta(1-40) inhibited the currents in a dose-dependent manner (0.1-10 microM) during treatment, gradually reversing after treatment. Amyloid-beta(1-40) and amyloid-beta(1-42) (0.1 microM) depressed alpha 4 beta 2 receptor currents to each 69% and 62% of original levels at 10-min treatment and lesser depression was obtained with alpha 7 receptors. Amyloid-beta(1-42) (0.1 microM) did not significantly inhibit AMPA receptor currents, but amyloid-beta(1-40) (0.1 microM) potentiated the currents to 145-191% of original levels. Amyloid-beta peptides, thus, exert their diverse actions on nicotinic ACh receptors and AMPA receptors, and the inhibitory actions on nicotinic ACh receptors may account for the deterioration of learning and memory in Alzheimer's disease.     

Activity of cytisine and its brominated isosteres on recombinant human alpha7, alpha4beta2 and alpha4beta4 nicotinic acetylcholine receptors

Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) alpha7-, alpha4beta2- and alpha4beta4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [alpha-(125)I]bungarotoxin ([alpha-(125)I]BgTx) to halpha7- and [(3)H]cy to halpha4beta2- or halpha4beta4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [alpha-(125)I]BgTx and [(3)H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at halpha7-nACh receptors but behaved as partial agonists at halpha4beta2- and halpha4beta4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at halpha7- and halpha4beta4-nACh receptors, but they elicited no responses on halpha4beta2-nACh receptors. Cy and 3-Br-cy produced dual dose-response curves (DRC) at both halpha4beta2- and halpha4beta4-nACh receptors, but ACh produced dual DRC only at halpha4beta2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing halpha4beta2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of halpha4beta2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.     

Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.

We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.     

Aporphine metho salts as neuronal nicotinic acetylcholine receptor blockers

(S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide (4) (vs [(3)H]cytisine) was the lowest at alpha4beta2 nAChR (K(i)=10 microM), and predicentrine methiodide (2) and xanthoplanine iodide (3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors (K(i) approximately 1 microM), while the affinity of the diphenolic boldine methiodide (1) was intermediate between these values. At homomeric alpha7 nAChR, xanthoplanine had the highest affinity (K(i)=10 microM) vs [(125)I]alpha-bungarotoxin while the other three compounds displaced the radioligand with K(i) values between 15 and 21 microM. At 100 microM, all four compounds inhibited the responses of these receptors to EC(50) concentrations of ACh. The effects of xanthoplanine iodide (3) were studied in more detail. Xanthoplanine fully inhibited the EC(50) ACh responses of both alpha7 and alpha4beta2 nACh receptors with estimated IC(50) values of 9+/-3 microM (alpha7) and 5+/-0.8 microM (alpha4beta2).     

The activation and inhibition of human nicotinic acetylcholine receptor by RJR-2403 indicate a selectivity for the alpha4beta2 receptor subtype

Human nicotinic acetylcholine (ACh) receptor subtypes expressed in Xenopus oocytes were characterized in terms of their activation by the experimental agonist RJR-2403. Responses to RJR-2403 were compared with those evoked by ACh and nicotine. These agonists were also characterized in terms of whether application of the drugs had the effect of producing a residual inhibition that was manifest as a decrease in subsequent control responses to ACh measured 5 min after the washout of the drug. For the activation of alpha4beta2 receptors, RJR-2403 had an efficacy equivalent to that of ACh and was more potent than ACh. RJR-2403 was less efficacious than ACh for other human receptor subtypes, suggesting that it is a partial agonist for all these receptors. Nicotine activated peak currents in human alpha4beta2 and alpha3beta2 receptors that were 85 and 50% of the respective ACh maximum responses. Nicotine was an efficacious activator of human alpha7 receptors, with a potency similar to ACh, whereas RJR-2403 had very low potency and efficacy for these receptors. At concentrations of <1 mM, RJR-2403 did not produce any residual inhibition of subsequent ACh responses for any receptor subtype. In contrast, nicotine produced profound residual inhibition of human alpha4beta2, alpha3beta2, and alpha7 receptors with IC(50) values of 150, 200, and 150 microM, respectively. Co-expression of the human alpha5 subunit with alpha3 and beta2 subunits had the effect of producing protracted responses to ACh and increasing residual inhibition by ACh and nicotine but not RJR-2403. In conclusion, our results, presented in the context of the complex pharmacology of nicotine for both activating and inhibiting neuronal nicotinic receptor subtypes, suggest that RJR-2403 will be a potent and relatively selective activator of human alpha4beta2 receptors.     

Alpha-conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells

The activity of alpha-conotoxin (alpha-CTX) ImI, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX ImI was a potent inhibitor of the neuronal nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 microM, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. Alpha-CTX ImI also inhibited nicotine-evoked 45Ca2+ uptake but not 45Ca2+ uptake stimulated by 56 mM K+. In contrast, alpha-CTX ImI had no effect at the neuromuscular junction over the concentration range 1-20 microM. Bovine chromaffin cells are known to contain the alpha3beta4, alpha7, and (possibly) alpha3beta4alpha5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha7 nicotinic receptors are not involved. We propose that alpha-CTX Iml interacts selectively with the functional (alpha3beta4 or alpha3beta4alpha5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.     

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.     

Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities.

The experiments described examine single channel currents recorded through Torpedo acetylcholine receptor channels stably expressed by a mouse fibroblast cell line. Closed-duration histograms were constructed from currents elicited by 0.5-300 microM acetylcholine (ACh). The concentration dependence of closed durations is well described by a four-state linear scheme with the addition of open-channel block by ACh. Analysis of closed durations measured at low concentrations gives estimates of the rate of opening of doubly liganded receptors, beta, the rate of dissociation of ACh from doubly liganded receptors, k-2, and the rate of channel closing, alpha. The rate of ACh dissociation from singly liganded receptors, k-1, is then deduced from closed-duration histograms obtained at intermediate ACh concentrations. With k-1, k-2 and beta determined, the rates of ACh association, k+1 and k+2, are estimated from fitting closed-duration histograms obtained over a range of high ACh concentrations. A complete set of rate constants is presented for three experimental conditions: (a) Ca2(+)-free extracellular solution containing 1 mM free Mg2+ at 22 degrees C, (b) Ca2(+)-free solution at 12 degrees C, and (c) extracellular Ca2+ and Mg2+, both at 0.5 mM, at 22 degrees C. For all three conditions the dissociation constant for the first agonist binding site is approximately 100-fold lower than that for the second site. The different affinities are due primarily to different dissociation rates. Both the association and dissociation rates depend strongly on temperature. At 22 degrees C ACh associates at diffusion-limited rates, whereas at 12 degrees C association is 30- to 60-fold slower. Also slowed at 12 degrees C are beta (4-fold), k-2 (3-fold), k-1 (25-fold), and alpha (15-fold). In contrast to the activation rate constants, those for ACh-induced block decrease only twofold between 22 and 12 degrees C. Changing from a Ca2(+)-free to a Ca2(+)-containing extracellular solution does not affect k+1 and k+2, but increases beta (twofold) and decreases k-2, k-1, and alpha (all twofold). Spectral analysis of single channel currents supports the parameter estimates obtained from fitting the open- and closed-duration histograms, and improves resolution of brief channel blockages produced by ACh.     

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E(rev)) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E(rev) of nicotine-induced current as a function of extracellular Na(+) concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K(+)/Na(+) permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca(2+) concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na(+), which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.     

The effects of beta3 subunit incorporation on the pharmacology and single channel properties of oocyte-expressed human alpha3beta4 neuronal nicotinic receptors

We compared the main properties of human recombinant alpha3beta4beta3 neuronal nicotinic receptors with those of alpha3beta4 receptors, expressed in Xenopus oocytes. beta3 incorporation decreased the channel mean open time (from 5.61 to 1.14 ms, after approximate correction for missed gaps) and burst length. There was also an increase in single channel slope conductance from 28.8 picosiemens (alpha3beta4) to 46.7 picosiemens (alpha3beta4beta3; in low divalent external solution). On the other hand, the calcium permeability (determined by a reversal potential method in chloride-depleted oocytes) and the pharmacological properties of beta3-containing receptors differed little from those of alpha3beta4. The main pharmacological difference in alpha3beta4beta3 "triplet" receptors was a 3-fold decrease in the potency of lobeline relative to acetylcholine. Nevertheless, there was no change in the rank order of potency for agonists (epibatidine > lobeline > cytisine, 1,1-dimethyl-4-phenylpiperazinium iodide, nicotine > acetylcholine > carbachol for both receptors; measured at low agonist concentrations). Sensitivity to the competitive antagonists trimetaphan (0.2-1 microM) and dihydro-beta-erythroidine (30 microM) was similar for the two combinations, with a Schild KB for trimetaphan of 76 and 66 nM on alpha3beta4 and alpha3beta4beta3, respectively. The change in single channel conductance confirms that beta3 replaces a beta4 subunit in the pentamer. The absence of pronounced differences in the pharmacological profile of the triplet receptor argues against a role for the beta3 subunit in the formation of agonist binding sites, whereas the changes in channel kinetics suggest an important effect on receptor gating. The shortening of the burst length of beta3-containing receptors implies that any synaptic currents mediated by such channels would have faster decay kinetics.     

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-β-erythroidine and α-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.     

Expression of functional nicotinic acetylcholine receptors in neuroepithelial bodies of neonatal hamster lung

Pulmonary neuroepithelial bodies (NEB) are presumed airway chemoreceptors involved in respiratory control, especially in the neonate. Nicotine is known to affect both lung development and control of breathing. We report expression of functional nicotinic acetylcholine receptors (nAChR) in NEB cells of neonatal hamster lung using a combination of morphological and electrophysiological techniques. Nonisotopic in situ hybridization method was used to localize mRNA for the beta 2-subunit of nAChR in NEB cells. Double-label immunofluorescence confirmed expression of alpha 4-, alpha 7-, and beta 2-subunits of nAChR in NEB cells. The electrophysiological characteristics of nAChR in NEB cells were studied using the whole cell patch-clamp technique on fresh lung slices. Application of nicotine ( approximately 0.1-100 microM) evoked inward currents that were concentration dependent (EC50 = 3.8 microM; Hill coefficient = 1.1). ACh (100 microM) and nicotine (50 microM) produced two types of currents. In most NEB cells, nicotine-induced currents had a single desensitizing component that was blocked by mecamylamine (50 microM) and dihydro-beta-erythroidine (50 microM). In some NEB cells, nicotine-induced current had two components, with fast- and slow-desensitizing kinetics. The fast component was selectively blocked by methyllcaconitine (MLA, 10 nM), whereas both components were inhibited by mecamylamine. Choline (0.5 mM) also induced an inward current that was abolished by 10 nM MLA. These studies suggest that NEB cells in neonatal hamster lung express functional heteromeric alpha 3 beta 2, alpha 4 beta 2, and alpha 7 nAChR and that cholinergic mechanisms could modulate NEB chemoreceptor function under normal and pathological conditions.     

Voltage-jump relaxation kinetics for wild-type and chimeric beta subunits of neuronal nicotinic receptors

We have studied the voltage-jump relaxation currents for a series of neuronal nicotinic acetylcholine receptors resulting from the coexpression of wild-type and chimeric beta 4/beta 2 subunits with alpha 3 subunits in Xenopus oocytes. With acetylcholine as the agonist, the wild-type alpha 3 beta 4 receptors displayed five- to eightfold slower voltage-jump relaxations than did the wild-type alpha 3 beta 2 receptors. In both cases, the relaxations could best be described by two exponential components of approximately equal amplitudes over a wide range of [ACh]'s. Relaxation rate constants increased with [ACh] and saturated at 20- to 30-fold lower concentrations for the alpha 3 beta 2 receptor than for the alpha 3 beta 4 receptor, as observed previously for the peak steady state conductance. Furthermore, the chimeric beta 4/beta 2 subunits showed a transition in the concentration dependence of the rate constants in the region between residues 94 and 109, analogous to our previous observation with steady state conductances. However, our experiments with a series of beta- subunit chimeras did not localize residues that govern the absolute value of the kinetic parameters. Hill coefficients for the relaxations also differed from those previously measured for steady state responses. The data reinforce previous conclusions that the region between residues 94 and 109 on the beta subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.     

A Novel Agonist Binding Site on Nicotinic Acetylcholine Receptors

Abstract

This report provides evidence that physostigmine (Phy) and benzoquinonium (BZQ) are able to activate nicotinic acetylcholine receptors (nAChRs) through binding site(s) distinct from those of the natural transmitter, ACh. Such findings are in agreement with a second pathway of activation of nAChRs. Receptor activation may be modulated through the novel site, and, consequently, physiological processes involving nicotinic synapses could be controlled. Using patch clamp techniques, single channel currents activated by ACh and anatoxin were recorded from frog interosseal muscle fibers under cell-attached condition and outside-out patches excised from cultured rat hippocampal neurons. Whole cell nicotinic currents were also studied in the cultured neurons. In most of the neurons, nicotinic responses were blocked by the nicotinic antagonists methyllycaconitine (MLA) and α-bungarotoxin (α-BGT). Evaluation of the effects of Phy and BZQ on the muscle and on the α-BGT- and MLA-sensitive neuronal nAChRs demonstrated that both compounds were open channel blockers at these receptors. Furthermore, at low micromolar concentrations, Phy and BZQ activated the nAChRs of all preparations tested, such an effect being unexpectedly resistant to α-BGT or MLA. Thus, the nAChRs could be activated via two distinct binding sites: one for ACh and the other for Phy and BZQ. These findings and previous biochemical results led us to suggest that a putative endogenous ligand could bind to the new site and thereby regulate the activation of nAChRs in nicotinic synapses.     

Suppression of the Nicotinic Acetylcholine Response in Rat Superior Cervical Ganglionic Neurons by Steroids

Abstract : The effects of various types of steroids on the nicotinic acetylcholine (ACh) receptor (nAChR)-mediated responses were investigated in superior cervical ganglionic neurons acutely dissociated from rats using nystatin perforated patch recording. ACh induced a peak followed by a gradual decrease in the inward current at a holding potential of -40 mV. Nicotine, but not muscarine, mimicked ACh. Hydrocortisone at a concentration of > 10^-6 M reversibly suppressed both the peak and steady-state nicotine-induced currents ( I _nic) in a noncompetitive manner. The inhibition of I _nic by hydrocortisone did not show any voltage dependency and persisted in the presence of either cyclic AMP modulators, forskolin and 3-isobutyl-1-methylxanthine, or a protein kinase A inhibitor, N -[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89). β-Estradiol, androsterone, aldosterone, and 17α-estradiol mimicked hydrocortisone in its inhibitory action on ACh-induced currents ( I _ACh). The potency for the inhibitory actions on I _Ach was as follows : androsterne > β-estradiol > hydrocortisone ≥ aldosterone =17α-estradiol. Cholesterol had no effect on the I _ACh. In conclusion, the structural characteristics of steroid are thus considered to be necessary to block nicotinic I _ACh in rat superior cervical ganglionic cells, whereas the cholesterol side chain might disturb the inhibitory action of the steroid skeleton on nAChRs.     

The mechanism of SR95531 inhibition at GABA receptors examined in human alpha1beta1 and alpha1beta1gamma2S receptors

We examined the interaction of GABA and the competitive inhibitor SR95531 at human alpha1beta1gamma2S and alpha1beta1 GABA(A) receptors expressed in Sf9 cells. The efficacy and potency of inhibition depended on the relative timing of the GABA and SR95531 applications. In saturating (10 mM) GABA, the half-inhibitory concentrations of SR95531 (IC50) when coapplied with GABA to alpha1beta1gamma2S or alpha1beta1 receptors were 49 and 210 microM for the peak and 18 and 130 microM for the plateau current, respectively. Our data are explained by an inhibition mechanism in which SR95531 and GABA bind to two sites on the receptor where the binding of GABA allows channel opening but SR95531 does not. The SR95531 affinity for both receptor types was approximately 200 nM and the binding rate was found to be 10-fold faster than that for GABA. The dual binding-site model gives insights into the differential effects of GABA and SR95531 on the peak and plateau currents. The model predicts the effect of SR95531 on GABA currents in the synapse (GABA concentration approximately mM) and at extrasynaptic (GABA concentration < or = microM) sites. The IC50 (50-100 nM) for the synaptic response to SR95531 was insensitive to the GABA affinity of the receptors whereas the IC50 (50-800 nM) for extrasynaptic inhibition correlated with the GABA affinity.     

Beta gamma dimers of G proteins inhibit atrial muscarinic K+ channels

It has been proposed that beta gamma dimers of signal-transducing G proteins mediate muscarinic activation of atrial K+ channels. We examined this hypothesis by testing the effects of beta gamma dimers from four sources (human erythrocytes, human placenta, bovine brain, and bovine retina) on single channel muscarinic K+ (K+[acetylcholine (ACh)]) currents in inside-out membrane patches of adult guinea pig atria. None of the four beta gamma dimer preparations stimulated K+[ACh] currents; on the contrary, each inhibited the currents whether the currents were activated with GTP alone (agonist-independent activity) or with GTP plus a muscarinic agonist (agonist-dependent activity). Detergents at concentrations used to suspend erythrocyte, brain, and placental beta gamma dimers had no effect by themselves, and detergents were not used with the retinal beta gamma dimers. We conclude that beta gamma dimers do not mediate stimulatory effects of the endogenous G protein that regulates the K+ channels. In fact beta gamma dimers appear to inhibit activation by the endogenous G alpha subunits. Further insight into the role of beta gamma dimers came from the observation that agonist-independent GTP-activated K+[ACh] currents were inhibited by beta gamma dimers at about one-tenth the concentration required to inhibit agonist-dependent activation. One possibility is that dimeric beta gamma may have a higher affinity for free alpha subunits than for alpha subunits associated with agonist-occupied receptors. Thus, in addition to the known requirement of beta gamma dimers for the interaction of alpha subunits with receptors, beta gamma dimers may also improve the signal-to-noise ratio for agonists by reducing agonist-independent background activities.     

Nicotine trapping causes the persistent desensitization of alpha4beta2 nicotinic receptors expressed in oocytes

To determine whether prolonged nicotine exposure persistently inactivates rat alpha4beta2 nicotinic receptors expressed in Xenopus oocytes, we measured the voltage-clamped alpha4beta2 response to acetylcholine (ACh) before and 24 h after, 1-h or 12-h incubations in 10 microm nicotine. A 12-h incubation in 10 microm nicotine depressed the alpha4beta2 ACh response for 24 h without affecting total or surface alpha4beta2 expression. To determine whether oocyte-mediated nicotine release caused this depression, we co-incubated an alpha4beta2-expressing oocyte with an un-injected one (pre-incubated in 10 microm nicotine for 12 h) for 24 h and measured the change in the alpha4beta2 ACh response. The response decreased by the same factor after the co-incubation as it did after a 12-h incubation in 10 microm nicotine and a 24-h incubation in nicotine-free media. Thus, oocyte-mediated nicotine release caused the persistent desensitization we observed after a 12-h incubation in 10 microm nicotine. Consistent with this result, measurements of [3H]nicotine release show that oocytes release enough nicotine into the wash media to desensitize alpha4beta2 receptors and that prolonged incubation in 300 microm ACh (which cannot readily cross the membrane or accumulate in acidic vesicles) did not persistently depress the alpha4beta2 response.