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Inhibition of nicotinic acetylcholine receptors by bicuculline
No full textA study was made on the effects of bicuculline, the classical γ-aminobutyric acid-A receptor antagonist, on heteromeric mouse muscle αβγδ, heteromeric neuronal rat α2β4 and α4β2 and homomeric human α7 nicotinic acetylcholine receptors (nAChRs), expressed in Xenopus oocytes. Bicuculline reduced the ACh-induced currents in a rapid and reversible way, with IC50 values of 34±1.5 μM for mouse muscle αβγδ and 12.4±0.7 and 18±1 μM for rat neuronal α2β4 and α4β2 nAChRs, respectively. Therefore, the three types of heteromeric receptors are inhibited by bicuculline but the neuronal α2β4 and α4β2 receptors were more sensitive than the muscle αβγδ receptor. The Hill coefficients for ACh-current inhibition were close to one for all types of receptors, suggesting a single site of action for bicuculline inhibition of nAChRs. Bicuculline shifted the ACh-dose-current response curve to the right and the maximal current was reduced, a reduction that for the heteromeric receptors was not overcome by high concentrations of ACh. The effect of bicuculline was examined at different membrane potentials, and the ACh-current-membrane potential relationships obtained indicate that the inhibition by bicuculline is voltage-dependent for muscle αβγδ and neuronal α2β4 and α4β2 nAChRs. All these results are consistent with the notion that bicuculline blocks the heteromeric muscle and neuronal nAChRs in a non-competitive way. Studies were also made on the wild type (wt α7) and mutant leu-to-threo (L248T) homomeric human neuronal α7-nAChRs. In sharp contrast to the heteromeric ACh receptors examined, bicuculline blocked in a competitive way the homomeric wt α7-nAChRs, as evidenced by a parallel shift of the bicuculline dose-ACh-current inhibition on raising the ACh concentration. Moreover, similar to the effects of serotonin on wt and mutant α7 ACh receptors, the mutation converted bicuculline from an antagonist into a competitive agonist. All this suggests that bicuculline may serve as a lead molecule to design new anticholinergic substances. © 2001 Published by Elsevier Science Ltd
Properties of neuronal alpha7 mutant nicotinic acetylcholine receptors gated by bicuculline.
No full textWe have shown previously that mutating to threonine the leucine residue in the M2 domain of the alpha7 nicotinic acetylcholine receptor (human L248T, L248T; chick L247T, L247T) converts bicuculline (BIC) from an antagonist into an agonist. In this work we studied the functional properties of the BIC-activated channels and report that, in Xenopus oocytes injected with L248T subunit cDNA, BIC activates single-channel currents that have similar conductances, but shorter mean burst duration, than the channels activated by ACh. In contrast, both the conductance and kinetics of the channels activated by either ACh or BIC are substantially the same in oocytes expressing L247T receptors. We have also shown previously that if Cys 189 and 190, which are thought to be at or near the transmitter binding site, are additionally mutated to Ser, the new mutant receptor (L247T-C189S-C190S) has a reduced affinity for ACh. We now find that the EC(50) in the BIC dose-current response relation, as well the characteristics of the channels activated by BIC, are similar in oocytes expressing either L247T or L247T-C189S-C190S receptors. On the other hand, ACh activation of L247T-C189S-C190S receptors gates channels whose mean open time and burst duration are much shorter than those of ACh-gated L247T-channels. Therefore, the gating kinetics of both L248T and L247R-C189S-C190S receptor-channels change when BIC is replaced by ACh; and we conclude that both ACh and BIC activate mutant alpha7 receptors with different patterns of activation
Arachidonic acid augments potassium outward currents in rat neocortical neurons in culture
No full textWE analysed the effects induced by arachidonic acid on voltage-dependent outward currents generated by rat neocortical neurones in culture in the presence of Na+ and Ca2+ channel blockers. These currents, recorded with whole-cell voltage-clamping techniques, were presumably carried by K+ and were characterized by an early transient and a late persistent component. Extracellular application of arachidonic acid (50 muM) enhanced both components of the voltage-dependent K+ response by 15-29% (n > 20 cells). These effects were reversible and not observed when a low dose (50 muM) of indomethacin was present in the bath (n = 7 cells). We conclude that one or more of the arachidonic acid metabolites of the cyclooxygenase pathway might be involved in the modulation of outward currents in neocortical cells in culture
Microtransplantation of cellular membranes from squid stellate ganglion reveals ionotropic GABA receptors
Full text linkAuthor Posting. © Marine Biological Laboratory, 2013. This article is posted here by permission of Marine Biological Laboratory for personal use, not for redistribution. The definitive version was published in Biological Bulletin 224 (2013): 47-52.The squid has been the most studied cephalopod, and it has served as a very useful model for investigating the events associated with nerve impulse generation and synaptic transmission. While the physiology of squid giant axons has been extensively studied, very little is known about the distribution and function of the neurotransmitters and receptors that mediate inhibitory transmission at the synapses. In this study we investigated whether γ-aminobutyric acid (GABA) activates neurotransmitter receptors in stellate ganglia membranes. To overcome the low abundance of GABA-like mRNAs in invertebrates and the low expression of GABA in cephalopods, we used a two-electrode voltage clamp technique to determine if Xenopus laevis oocytes injected with cell membranes from squid stellate ganglia responded to GABA. Using this method, membrane patches containing proteins and ion channels from the squid's stellate ganglion were incorporated into the surface of oocytes. We demonstrated that GABA activates membrane receptors in cellular membranes isolated from squid stellate ganglia. Using the same approach, we were able to record native glutamate-evoked currents. The squid's GABA receptors showed an EC50 of 98 μmol l–1 to GABA and were inhibited by zinc (IC50 = 356 μmol l–1). Interestingly, GABA receptors from the squid were only partially blocked by bicuculline. These results indicate that the microtransplantation of native cell membranes is useful to identify and characterize scarce membrane proteins. Moreover, our data also support the role of GABA as an ionotropic neurotransmitter in cephalopods, acting through chloride-permeable membrane receptors.Grass Foundation Fellowships to
L.C. and A.L. (www.grassfoundation.org). L.C. was additionally
supported by the Ph.D. in Neurophysiology program
of the University of Rome “La Sapienza.” All authors
were Grass Fellows. This work was supported by Ministero
della Sanita` Antidoping and PRIN project 2009 (to E.P.)
The desensitization of the embryonic mouse muscle acetylcholine receptor depends on the cellular environment.
No full textThe rate of desensitization of nicotinic acetylcholine (ACh) receptor (nAChR), an important characteristic of nAChR function, was studied in myotubes of the mouse C2C12 cell line at different times after fusion, by measuring the decay of ACh-evoked currents (IACh) under various patch-clamp configurations. We observed a progressive slowing of IACh decay rate (half-decay time rose from about 0.5 s to over 5 s) in myotubes of increasing size (i.e. age) under all experimental conditions, except in outside-out patches, when IACh decayed as fast as in the smallest myotubes. Single-channel conductance (about 35 pS) and open time (about 3.5 ms), measured in outside-out and cell-attached patches, were independent of myotube size. In Xenopus oocytes injected with poly(A+)RNA extracted from C2C12 myoblasts or mature myotubes, IACh decay was about 50 times slower than in myotubes. Neither cAMP-dependent nor diacylglycerol-dependent protein kinases, actin nor microtubule polymerization state influenced IACh decay. Our data indicate that the cellular environment, but not readily dialysable cytosolic factors, markedly influences the functional behaviour of nAChR
The single-channel properties of human acetylcholine alpha 7 receptors are altered by fusing alpha 7 to the green fluorescent protein
No full textNeuronal nicotinic acetylcholine (AcCho) receptors composed of alpha7-subunits (alpha7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their single-channel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) alpha7-AcChoRs, we identified two classes of channel conductance: a low conductance (gamma(L)) of 72 pS and a high one (gamma(H)) of 87 pS, with mean open-times (tau(op)) of 0.6 ms. The same classes of conductances, but longer tau(op) (3 ms), were seen in experiments with chimeric alpha7 receptors in which the wtalpha7 extracellular C terminus was fused to the green fluorescent protein (wtalpha7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing alpha7 receptors carrying a Leu-to-Thr 248 mutation (mutalpha7) or oocytes expressing chimeric mutalpha7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wtalpha7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter tau(op), than those activated by AcCho. Accordingly, human homomeric wtalpha7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mutalpha7 receptors open channels of lower conductance and longer lifetime than those gated by wtalpha7-AcChoRs, and these parameters are not greatly altered by fusing the mutalpha7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function
Threonine-for-leucine mutation within domain M2 of the neuronal a7 nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist
No full textA study was made of the effects of 5-hydroxy-tryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (I-AcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 mu M and a Hill coefficient (n(H)) of 1.2. The inhibition of I-AcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism, 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA, In contrast, 5HT elicited membrane currents (I-5HT) in oocytes injected with cDNA encoding an cv mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)), I-5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 mu M). Furthermore, the characteristics of I-5HT, including its voltage dependence, were similar to those of I-AcCho. The 5HT dose-I-5HT response gave an apparent dissociation constant EC(50) of 23.5 mu M and a Hill coefficient n(H) of 1.7, which were not modified by the presence of AcCho, Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2
Modulation of voltage-activated channels by calcitonini gene-related peptide in cultured rat neurones
No full text1. Whole-cell currents were recorded from cultures of dissociated neocortical neurones of the rat. Rat alpha-calcitonin gene-related peptide (CGRP; 1 nM-1-mu-M) caused significant dose-dependent decreases in the voltage-activated transient (A-current) and delayed rectifier K+ currents. Forskolin (10 nM-20-mu-M) mimicked this effect. Peak K+ currents were gradually decreased after loading neurones with cyclic AMP (100-mu-M) through patch pipettes. CGRP was ineffective in neurones loaded with cyclic AMP.
2. CGRP (0.5-2 mu-M) increased cytosolic cyclic AMP concentration and this effect was mimicked by forskolin (5-40 mu-M).
3. CGRP (0.1-1 mu-M) reduced high-threshold Ca2+ currents; as did forskolin (5-20 mu-M) and cyclic AMP loaded into the neurones. In contrast, low-threshold Ca2+ currents were not affected by any of these agents.
4. Voltage-activated Na+ currents were significantly reduced by both CGRP (0.1-1 mu-M) and forskolin (5-20 mu-M). A similar effect was observed when cells were loaded with cyclic AMP.
5. We conclude that, in neocortical neurones, CGRP attenuates voltage-activated currents by stimulating the intracellular cyclic AMP signalling system
Strychnine activates neuronal a7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains
No full textRecent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric alpha 7 receptors, and that mutating Leu-247 of the alpha 7 nicotinic AcCho receptor-channel domain (L247T alpha 7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type alpha 7 or L247T alpha 7 receptors, In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247T alpha 7 receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189-190S alpha 7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker a-bungarotoxin, Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic alpha 7 receptors, After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1, A pentameric symmetrical model yields the striking conclusion that two identical alpha 7 subunits may be sufficient to determine the functional properties of alpha 7 receptors
Neuronal nicotinic threonine-for-leucine 247 alpha7 mutant receptors show different gating kinetics
No full textMutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric alpha7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the alpha7 mutant receptor for 5HT is not modified by the presence of dihydro-beta-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates alpha7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system
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