Heterogeneity of neuronal nicotinic acetylcholine receptors: Structural and functional aspects

Decay kinetics of the postsynaptic excitatory currents (EPSC), distribution of the antibodies specific to different α-subunits of neuronal nicotinic acetylcholine receptors (nAChR), and the effects of these antibodies on ACh-induced membrane currents were studied in neurons of different autonomic ganglia of rats. It was shown that α3-, α5- and α7-subunits were present in all studied cultured neurons of the rat superior cervical ganglion (SCG), while the α4-subunit was present only in about half of the neurons; this α-subunit distribution differed from that in cultured intracardial neurons of rats. Two nAChR populations were found in rat SCG neurons, and a series of nAChR populations were found in murine superior mesenteric ganglion neurons; they differed in kinetics of their ion channel activity, voltage dependence and the rate of their open channel blockade. The possible functional role of neuronal nAChR heterogeneity is discussed.     

Structural domains of agrin required for clustering of nicotinic acetylcholine receptors.

Agrin is an extracellular matrix component which promotes the clustering of nicotinic acetylcholine receptors (nAChRs) and other proteins at the neuromuscular junction. This aggregation process is one of the earliest steps in synapse formation. Expression of highly active isoforms of agrin, generated by alternative splicing, is restricted to neurons in the central nervous system (CNS) including motoneurons. In the experiments reported here we investigate the regions of agrin necessary for nAChR clustering activity using two different methods. First, we expressed truncated soluble forms of the agrin protein in mammalian cells and assessed their clustering activity. Second, we generated a panel of monoclonal antibodies (mAbs) against agrin and mapped their epitopes. Several mAbs block agrin-induced aggregation of nAChRs. One of the mAbs, Agr86, binds exclusively to the CNS-specific splicing variants and thus identifies an epitope common only to these more active isoforms. Mapping of the Agr86 epitope suggests that alternative splicing results in a distributed conformational change in the agrin protein. Taken together our data suggest that four domains in the C-terminal 55 kDa of agrin contribute to its nAChR clustering activity.     

Protein phosphorylation of nicotinic acetylcholine receptors

The nicotinic acetylcholine receptor (nAcChR) is a ligand-gated ion channel found in the postsynaptic membranes of electric organs, at the neuromuscular junction, and at nicotinic cholinergic synapses of the mammalian central and peripheral nervous system. The nAcChR from Torpedo electric organ and mammalian muscle is the most well-characterized neurotransmitter receptor in biology. It has been shown to be comprised of five homologous (two identicle) protein subunits (alpha 2 beta gamma delta) that form both the ion channel and the neurotransmitter receptor. The nAcChR has been purified and reconstituted into lipid vesicles with retention of ion channel function and the primary structure of all four protein subunits has been determined. Protein phosphorylation is a major posttranslational modification known to regulate protein function. The Torpedo nAcChR was first shown to be regulated by phosphorylation by the discovery that postsynaptic membranes contain protein kinases that phosphorylate the nAcChR. Phosphorylation of the nAcChR has since been shown to be regulated by the cAMP-dependent protein kinase, protein kinase C, and a tyrosine-specific protein kinase. Phosphorylation of the nAcChR by cAMP-dependent protein kinase has been shown to increase the rate of nAcChR desensitization, the process by which the nAcChR becomes inactivated in the continued presence of agonist. In cultured muscle cells, phosphorylation of the nAcChR has been shown to be regulated by cAMP-dependent protein kinase, a Ca2+-sensitive protein kinase, and a tyrosine-specific protein kinase. Stimulation of the cAMP-dependent protein kinase in muscle also increases the rate of nAcChR desensitization and correlates well with the increase in nAcChR phosphorylation. The AcChR represents a model system for how receptors and ion channels are regulated by second messengers and protein phosphorylation.     

Structure-function relationships in nicotinic acetylcholine receptors

1. A combination of molecular, biochemical, electrophysiological and immunological approaches has begun to resolve some of the questions about structure-function relationships of nicotinic acetylcholine receptors (AchRs). 2. Current structural studies suggest that models of the subunits which propose four transmembrane domains are correct. 3. It is also probable that the carboxy termini of the subunits are extracellular, while the putative amphpathic helix is intracellular. 4. Electrophysiological and ligand-binding experiments suggest that the M2 region forms the wall of the ion channel. 5. We have isolated clones from PC12 and rat brain cDNA libraries which we have shown, by functional expression, code for members of a gene family of nicotinic acetylcholine receptor subunits. 6. In situ hybridization studies have shown that the neuronal receptor subunit mRNAs are expressed in the mammalian central nervous system. 7. The muscle and neuronal nicotinic AchR subtypes we have expressed show differences in their pharmacological properties. 8. The isolation and identification of clones which code for receptors and voltage-activated ion channels will help in the understanding of a variety of disease states and assist in the design of drugs which are specific for unique molecular targets.     

Emerging structure of the nicotinic acetylcholine receptors

The conversion of acetylcholine binding into ion conduction across the membrane is becoming more clearly understood in terms of the structure of the receptor and its transitions. A high-resolution structure of a protein that is homologous to the extracellular domain of the receptor has revealed the binding sites and subunit interfaces in great detail. Although the structures of the membrane and cytoplasmic domains are less well determined, the channel lining and the determinants of selectivity have been mapped. The location and structure of the gates, and the coupling between binding sites and gates, remain to be established.     

Activation of skeletal muscle nicotinic acetylcholine receptors

Summary and Conclusions Work over the past ten years has greatly increased our understanding of both the structure and function of the muscle nicotinic acetylcholine receptor. There is a strongly supported general picture of how the receptor functions: agonist binds rapidly to sites of low affinity and channel opening occurs at a rate comparable to the agonist dissociation rate. Channel closing is slow, so the channel has a high probability of being open if both agonist-binding sites are occupied by ACh. Results of expression studies have shown that each subunit can influence AChR activation and have given a structural basis for the major physiological change known for muscle AChR, the developmental change in AChR activation. These general statements notwithstanding, there are still major areas of uncertainty which limit our understanding. We have emphasized these areas of uncertainty in this review, to indicate what needs to be done.First, the quantitative estimates of rate constants are not as strongly supported as they should be. The major reasons are twofold—uncertainties about the interpretation of components in the kinetic data and difficulties of resolving brief events. As a result, any inferences about the functional consequences of structural alterations must remain tenuous.Second, the functional behavior of individual AChRs is not as well understood as it should be. The kinetic behavior of an individual receptor clearly can be complex (section II). In addition, there is evidence that superimposed on this complexity there may be stable and kinetically distinguishable populations of receptors (section III). Until the basis for the kinetically defined populations is clarified, kinetic parameters for receptors of defined structure cannot be unambiguously obtained.Finally, it is not surprising that the studies of AChR of altered structure have not given definitive results. Two reasons should be apparent from the preceding points: there is not a fully supported approach for kinetic analysis, and the normal population may not be clearly defined. An additional complication is also emerging, in that the available data support the idea that specific residues distributed over all subunits may influence AChR activation. This possibility renders the task of analysis that much more difficult.The muscle nicotinic AChR has served as a prototype for the family of transmitter-gated membrane channels, which includes the muscle and neuronal nicotinic receptors, the GABA_A, the glycine and possibly the non-NMDA excitatory amino acid receptor (Stroud et al., 1990). It is interesting to note that the functional properties of the GABA_A receptor, probably the best-studied of the other members of the family are rather similar. In particular, opentime and burst durations show multiple components interpreted as reflecting openings of singly and doubly liganded receptors (Mathers & Wang, 1988; Macdonald et al., 1989), the distribution of gaps indicates a relatively complex gating scheme (Twyman et al., 1990; Weiss & Magleby, 1989), and multiple kinetic modes are likely to exist (Newland et al., 1991). The situation with regards to the effects of GABA_A receptor subunit stoichiometry is more complex than for muscle AChR (e.g., Luddens & Wisden, 1991), perhaps similar to that found for neuronal nicotinic AChR (Papke et al., 1989; Luetje et al., 1990; Luetje & Patrick, 1991). Overall, it appears that the unresolved questions about the muscle nicotinic AChR are not indications that this is an exceptionally complicated transmitter-gated channel. Rather, it appears to be a relatively straightforward member of the family, and the lessons we learn from studying it are likely to be directly applicable to other receptors.We thank many friends for discussion, including Tony Auerbach, Paul Brehm, Jim Dilger, Meyer Jackson, and Chuck Stevens who told us about data before publication. Research in the authors' laboratories is supported by grants from the NIH (CL and JHS) and the AHA (CL).     

Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.     

Role of non-neuronal nicotinic acetylcholine receptors in angiogenesis

Angiogenesis is a critical physiological process for cell survival and development. Endothelial cells, necessary for the course of angiogenesis, express several non-neuronal nicotinic acetylcholine receptors (AChRs). The most important functional non-neuronal AChRs are homomeric α7 AChRs and several heteromeric AChRs formed by a combination of α3, α5, β2, and β4 subunits, including α3β4-containing AChRs. In endothelial cells, α7 AChR stimulation indirectly triggers the activation of the integrin αvβ3 receptor and an intracellular MAP kinase (ERK) pathway that mediates angiogenesis. Non-selective cholinergic agonists such as nicotine have been shown to induce angiogenesis, enhancing tumor progression. Moreover, α7 AChR selective antagonists such as α-bungarotoxin and methyllycaconitine as well as the non-specific antagonist mecamylamine have been shown to inhibit endothelial cell proliferation and ultimately blood vessel formation. Exploitation of such pharmacologic properties can lead to the discovery of new specific cholinergic antagonists as anti-cancer therapies. Conversely, the pro-angiogenic effect elicited by specific agonists can be used to treat diseases that respond to revascularization such as diabetic ischemia and atherosclerosis, as well as to accelerate wound healing. In this mini-review we discuss the pharmacological evidence supporting the importance of non-neuronal AChRs in angiogenesis. We also explore potential intracellular mechanisms by which α7 AChR activation mediates this vital cellular process.     

Ca2+ permeability of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are expressed in muscle cells and neurons, as well as in an increasing number of other cell types. The nAChR channels are permeable to cations, including Ca(2+). Ca(2+) entry through nAChR channels has been shown to modulate several Ca(2+)-dependent cellular processes, such as neurotransmitter release, synaptic plasticity, and cell motility. The value of Ca(2+) permeability associated to a particular nAChR subtype thus represents an important indication for its physiological role. This review summarizes the quantitative data on Ca(2+) permeability obtained from several nAChR subtypes in native and heterologous systems. Different experimental approaches are compared, and the structural determinants of Ca(2+) permeability are discussed.     

Stimulation of nicotinic acetylcholine receptors protects motor neurons

The present study demonstrated that administration of nicotine prevented glutamate-induced motor neuronal death in primary cultures of the rat spinal cord. The nicotine-induced neuroprotection was inhibited by either dihydro-beta-erythroidin (DHbetaE) or alpha-bungarotoxin (alphaBT), suggesting that it is mediated through both alpha4beta2 and alpha7 nicotinic acetylcholine receptors (nAChRs). Both alpha4beta2 and alpha7 nAChRs were identified on rat spinal motor neurons by immunohistochemical methods. We also demonstrated that galantamine, an acetylcholinesterase inhibitor with allosteric nAChR-potentiating ligand properties, prevented glutamate-induced motor neuronal death. These results suggest that stimulation of nAChR may be used as a treatment for ALS.     

Neosurugatoxin blocks nicotinic acetylcholine receptors in the brain

Neosurugatoxin, a neurotoxin isolated from the Japanese ivory mollusc ($\text{Babylonia japonica}$) is a nicotinic antagonist with a specificity towards ganglionic nicotinic receptors. At low concentration (5 × 10^?8 M) neosurugatoxin inhibited the release of [³H]dopamine evoked by 1,1-dimethyl-4-phenylpiperazinium (DMPP) from rat striatal nerve terminals, without affecting the response to K⁺-depolarisation. In contrast, αbungarotoxin did not antagonise the action of DMPP. Neosurugatoxin also inhibited [³H] nicotine binding to rat brain membranes but had no effect on [¹²⁵I]αbungarotoxin binding to the same tissue preparation. These results support the view that functional nicotinic receptors in the CNS resemble ganglionic nicotinic receptors. Neosurugatoxin has considerable potential as a useful probe for such receptors in the brain.     

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.     

Structural model of nicotinic acetylcholine receptor isotypes bound to acetylcholine and nicotine

Background

Nicotine is a psychoactive drug presenting a diverse array of biological activities, some positive, such as enhancement of cognitive performances, others negative, such as addiction liability. Ligands that discriminate between the different isotypes of nicotinic acetylcholine receptors (nAChRs) could present improved pharmacology and toxicity profile.

Results

Based on the recent crystal structure of a soluble acetylcholine binding protein from snails, we have built atomic models of acetylcholine and nicotine bound to the pocket of four different human nAChR subtypes. The structures of the docked ligands correlate with available biochemical data, and reveal that the determinants for isotype selectivity are relying essentially on four residues, providing diversity of the ligand binding pocket both in terms of Van der Waals boundary, and electrostatic potential. We used our models to screen in silico a large compound database and identify a new ligand candidate that could display subtype selectivity.

Conclusion

The nAChR-agonist models should be useful for the design of nAChR agonists with diverse specificity profiles.     

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12′ position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12′ lysines in the α, γ and δ subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK _a values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in β or ε subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK _a values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12′ lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of α subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channel.     

Expression of nicotinic acetylcholine receptors in aneural Xenopus embryos

During gastrulation in vertebrate embryos, the mesoderm moves inward and under the ectoderm and these two cell layers subsequently differentiate in close proximity to each other, providing an opportunity for the exchange of inductive signals. This study examines whether the activation of muscle nicotinic acetylcholine receptor (AChR) genes and the subsequent expression of receptors in Xenopus myotomal muscle are dependent on interaction between the ectoderm and the mesoderm, or their derivatives, after the onset of gastrulation. We eliminated such interaction by inducing total exogastrulation of Xenopus embryos. During exogastrulation, the mesoderm moves away from the ectoderm, and the nervous system fails to develop. Single channel recordings from the myotomal muscle of exogastrulated embryos revealed the presence of two major classes of AChRs, which could be distinguished on the basis of channel conductance. The current amplitudes, conductances, reversal potentials, and open times of these channels closely resembled those reported for the two major classes of AChR channels normally expressed in vivo. We conclude that interaction between ectoderm and mesoderm following the onset of gastrulation is not required for the future expression of the major classes of AChRs in myotomal muscle.     

alpha-Conotoxins, small peptide probes of nicotinic acetylcholine receptors

alpha-Conotoxins, a family of small peptides from the venoms of the Conus marine moluscs, are selective, snake alpha-neurotoxin-competitive antagonists of the nicotinic acetylcholine receptor. A new alpha-conotoxin, SIA, has been purified, sequenced, and synthesized. Cross-linking with bivalent reagents and photoaffinity labeling of the acetylcholine receptor with alpha-conotoxin yield covalent adducts. Surprisingly, cross-linking to other subunits is considerably more efficient than to the alpha subunit. The relative efficiency of photoactivatable cross-linking to different subunits of the receptor is a function of placement of the photoactivatable group on the toxin. Since the structures of alpha-conotoxins can be solved by 2D NMR [see Pardi et al. (1989) Biochemistry 28, 5494-5508; Kobayashi et al. (1989) Biochemistry 28, 4853-4860], this family of toxins should provide a set of new ligands for probing the acetylcholine receptor with considerable precision.     

Cloning and expression of zebrafish neuronal nicotinic acetylcholine receptors

We propose to use the zebrafish (Danio rerio) as a vertebrate model to study the role of neuronal nicotinic acetylcholine receptors (nAChR) in development. As a first step toward using zebrafish as a model, we cloned three zebrafish cDNAs with a high degree of sequence similarity to nAChR beta3, alpha2 and alpha7 subunits expressed in other species. RT-PCR was used to show that the beta3 and alpha2 subunit RNAs were present in zebrafish embryos only 2-5hours post-fertilization (hpf) while alpha7 subunit RNA was not detected until 8hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish beta3, alpha2, and alpha7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity [3H]-epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC(50) values of 28.6pM and 29.7nM and in 5dpf embryos with IC(50) values of 28.4pM and 8.9nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.     

Blocking of neuronal nicotinic acetylcholine receptors with d-sparteine derivatives

The effects of d-sparteine (d-SP) and its two derivatives, N-methylsparteine (IEM-1820) and N-phenylsparteine (IEM-1821), on nicotinic acetylcholine receptors (nAChR) of the rat superior cervical ganglion neurons were studied. Membrane currents evoked by iontophoretically applied acetylcholine were recorded using the patch-clamp recording technique in the whole-cell configuration. All three compounds were found to block nAChR competitively, the blocking activity being increased with an increase in the size of the blocking molecule. The EC₅₀ values for d-SP, IEM-1820, and IEM-1821 were equal to 2.06±0.38 µM (n=3), 1.64±0.41 µM (n=4), and 0.65±0.17 µM (n=3), respectively. It was assumed that the increase in efficiency of blocking is related to the decrease in the rate of dissociation of the blocker and receptor molecules.Neirofiziologiya/Neurophysiology, Vol. 26, No. 4, pp. 266–269, July–August, 1994.