Modulation of nicotinic acetylcholine receptors by strychnine

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex.

Neuronal nicotinic threonine-for-leucine 247 α7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric α7 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 α7 mutant receptor for 5HT is not modified by the presence of dihydro-β-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 α7 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.

Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains

Recent 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 α7 receptors, and that mutating Leu-247 of the α7 nicotinic AcCho receptor-channel domain (L247Tα7; 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 α7 or L247Tα7 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 L247Tα7 receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189–190Sα7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker α-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 α7 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 α7 subunits may be sufficient to determine the functional properties of α7 receptors.

Point mutant mice with hypersensitive α4 nicotinic receptors show dopaminergic deficits and increased anxiety

Knock-in mice were generated that harbored a leucine-to-serine mutation in the α4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.

β-Amyloid peptide blocks the response of α7-containing nicotinic receptors on hippocampal neurons

Alzheimer's disease produces a devastating decline in mental function, with profound effects on learning and memory. Early consequences of the disease include the specific loss of cholinergic neurons in brain, diminished cholinergic signaling, and the accumulation of β-amyloid peptide in neuritic plaques. Of the nicotinic acetylcholine receptors at risk, the most critical may be those containing the α7 gene product (α7-nAChRs), because they are widespread, have a high relative permeability to calcium, and regulate numerous cellular events in the nervous system. With the use of whole-cell patch–clamp recording we show here that nanomolar concentrations of β-amyloid peptides specifically and reversibly block α7-nAChRs on rat hippocampal neurons in culture. The block is noncompetitive, voltage-independent, and use-independent and is mediated through the N-terminal extracellular domain of the receptor. It does not appear to require either calcium influx or G protein activation. β-Amyloid blockade is likely to be a common feature of α7-nAChRs because it applies to the receptors at both somato-dendritic and presynaptic locations on rat hippocampal neurons and extends to homologous receptors on chick ciliary ganglion neurons as well. Because α7-nAChRs in the central nervous system are thought to have numerous functions and recently have been implicated in learning and memory, impaired receptor function in this case may contribute to cognitive deficits associated with Alzheimer's disease.

A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors.

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of changes that activate a cation-selective conductance. By measuring electrophysiological responses in chimeric alpha7/alpha3 receptors expressed in Xenopus oocytes, we have showed the involvement of the M2-M3 loop in coupling agonist binding to the channel gate. An aspartate residue therein, Asp-266 in the alpha7 subunit, was identified by site-directed mutagenesis as crucial, since mutants at this position exhibited very poor functional responses to three different nicotinic agonists. We have extended this investigation to another neuronal nicotinic receptor (alpha3/beta4), and found that a homologous residue in the beta4 subunit, Asp-268, played a similar role in coupling. These findings are consistent with a hypothesis that the aspartate residue in the M2-M3 loop, which is conserved in all homomer-forming alpha-type subunits and all neuronal beta-type subunits that combine to form functional receptors, is a major determinant of information transmission from binding site to channel gate in all neuronal nicotinic receptors.

Activation of nicotinic receptor-induced postsynaptic responses to luteinizing hormone-releasing hormone in bullfrog sympathetic ganglia via a Na+-dependent mechanism

Nicotine at very low doses (5–30 nM) induced large amounts of luteinizing hormone-releasing hormone (LHRH) release, which was monitored as slow membrane depolarizations in the ganglionic neurons of bullfrog sympathetic ganglia. A nicotinic antagonist, d-tubocurarine chloride, completely and reversibly blocked the nicotine-induced LHRH release, but it did not block the nerve-firing-evoked LHRH release. Thus, nicotine activated nicotinic acetylcholine receptors and produced LHRH release via a mechanism that is different from the mechanism for evoked release. Moreover, this release was not caused by Ca2+ influx through either the nicotinic receptors or the voltage-gated Ca2+ channels because the release was increased moderately when the extracellular solution was changed into a Ca2+-free solution that also contained Mg2+ (4 mM) and Cd2+ (200 μM). The release did not depend on Ca2+ release from the intraterminal Ca2+ stores either because fura-2 fluorimetry showed extremely low Ca2+ elevation (≈30 nM) in response to nicotine (30 nM). Moreover, nicotine evoked LHRH release when [Ca2+] elevation in the terminals was prevented by loading the terminals with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid and fura-2. Instead, the nicotine-induced release required extracellular Na+ because substitution of extracellular NaCl with N-methyl-d-glucamine chloride completely blocked the release. The Na+-dependent mechanism was not via Na+ influx through the voltage-gated Na+ channels because the release was not affected by tetrodotoxin (1–50 μM) plus Cd2+ (200 μM). Thus, nicotine at very low concentrations induced LHRH release via a Na+-dependent, Ca2+-independent mechanism.

Galantamine: Effect on nicotinic receptor binding, acetylcholinesterase inhibition, and learning

Classical eyeblink conditioning is a well-characterized model paradigm that engages the septohippocampal cholinergic system. This form of associative learning is impaired in normal aging and severely disrupted in Alzheimer's disease (AD). Some nicotinic cholinergic receptor subtypes are lost in AD, making the use of nicotinic allosterically potentiating ligands a promising therapeutic strategy. The allosterically potentiating ligand galantamine (Gal) modulates nicotinic cholinergic receptors to increase acetylcholine release as well as acting as an acetylcholinesterase (AChE) inhibitor. Gal was tested in two preclinical experiments. In Experiment 1 with 16 young and 16 older rabbits, Gal (3.0 mg/kg) was administered for 15 days during conditioning, and the drug significantly improved learning, reduced AChE levels, and increased nicotinic receptor binding. In Experiment 2, 53 retired breeder rabbits were tested over a 15-wk period in four conditions. Groups of rabbits received 0.0 (vehicle), 1.0, or 3.0 mg/kg Gal for the entire 15-wk period or 3.0 mg/kg Gal for 15 days and vehicle for the remainder of the experiment. Fifteen daily conditioning sessions and subsequent retention and relearning assessments were spaced at 1-month intervals. The dose of 3.0 mg/kg Gal ameliorated learning deficits significantly during acquisition and retention in the group receiving 3.0 mg/kg Gal continuously. Nicotinic receptor binding was significantly increased in rabbits treated for 15 days with 3.0 mg/kg Gal, and all Gal-treated rabbits had lower levels of brain AChE. The efficacy of Gal in a learning paradigm severely impaired in AD is consistent with outcomes in clinical studies.

Tonic nicotinic modulation of serotoninergic transmission in the spinal cord

The spinal serotoninergic projection from the raphe magnus has been shown to modulate nociceptive inputs, and activation of this projection mediates nicotine-elicited analgesia. Here, we investigate the interactions between cholinergic and serotoninergic systems in the spinal cord, by conducting serotonin [5-hydroxytryptamine (5-HT)] efflux experiments on mouse spinal slices. At least three spinal populations of nicotinic receptors are distinguished that affect 5-HT release. The first could be directly located on serotoninergic terminals, is insensitive to nanomolar concentrations of methyllicaconitine (MLA), and may be subjected to a basal (not maximal) cholinergic tone. The second is tonically and maximally activated by endogenous acetylcholine, insensitive to nanomolar concentrations of MLA, and present on inhibitory neurons. The last is also present on inhibitory neurons but is sensitive to nanomolar concentrations of MLA and not tonically activated by acetylcholine. Multiple nicotinic acetylcholine receptor populations thus differentially exert tonic or not tonic control on 5-HT transmission in the spinal cord. These receptors may be major targets for nicotine effects on antinociception. In addition, the presence of a tonic nicotinic modulation of 5-HT release indicates that endogenous acetylcholine plays a role in the physiological regulation of descending 5-HT pathways to the spinal cord.

Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores

In this report we provide evidence that neuronal nicotinic acetylcholine receptors (nAChRs) are present on hippocampal astrocytes and their activation produces rapid currents and calcium transients. Our data indicate that these responses obtained from astrocytes are primarily mediated by an AChR subtype that is functionally blocked by α-bungarotoxin (αBgt) and contains the α7 subunit (αBgt-AChRs). Furthermore, their action is unusual in that they effectively increase intracellular free calcium concentrations by activating calcium-induced calcium release from intracellular stores, triggered by influx through the receptor channels. These results reveal a mechanism by which αBgt-AChRs on astrocytes can efficiently modulate calcium signaling in the central nervous system in a manner distinct from that observed with these receptors on neurons.

Threonine-for-leucine mutation within domain M2 of the neuronal alpha(7) nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist.

A study was made of the effects of 5-hydroxytryptamine (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 (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho 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 (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH 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.

Quantal release at a neuronal nicotinic synapse from rat adrenal gland.

The neuronal nicotinic synapse in tissue slices of the adrenal medulla was studied with whole-cell patch-clamp. Excitatory postsynaptic currents (EPSCs) were evoked by local field stimulation or occurred spontaneously especially when external [K+] was increased. EPSCs were carried by channels sharing biophysical and pharmacological properties of neuronal-type nicotinic receptors (nAChRs). A single-channel conductance (gamma) of 43-45 pS was found from nonstationary variance analysis of EPSCs. Spontaneous EPSCs were tetrodotoxin-insensitive and Ca(2+)-dependent and occurred in burst-like clusters. Quantal analysis of spontaneous EPSCs gave a quantal size of 20 pA and amplitude histograms were well described by binomial models with low values of quantal content, consistent with a small number of spontaneously active release sites. However, rare large amplitude EPSCs suggest that the total number of sites is higher and that extrajunctional receptors are involved. Our estimates of quantal content and size at the chromaffin cell neuronal nicotinic synapse may be useful in characterizing central neuronal-type nicotinic receptor-mediated cholinergic synaptic transmission.

Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.

Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.

Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins.