On the nature of partial agonism in the nicotinic receptor superfamily (original) (raw)
Related papers
Activation of a nicotinic acetylcholine receptor
Biophysical Journal, 1984
We studied activation of the nicotinic acetylcholione (ACh) receptor on cells of a mouse clonal muscle cell line (BC3HI). We analyzed single-channel currents through outside-out patches elicited with various concentrations of acetylcholine (ACh), carbamylcholine (Carb) and suberyldicholine (Sub). Our goal is to determine a likely reaction scheme for receptor activation by agonist and to determine values of rate constants for transitions in that scheme. Over a wide range of agonist concentrations the open-time duration histograms are not described by single exponential functions, but are well-described by the sum of two exponentials, a brief-duration and a long-duration component. At high concentration, channel openings occur in groups and these groups contain an excess number of brief openings. We conclude that there are two open states of the ACh receptor with different mean open times and that a single receptor may open to either open state. The concentration dependence of the numbers of brief and long openings indicates that brief openings do not result from the opening of channels of receptors which have only one agonist molecule bound to them. Closed-time duration histograms exhibit a major brief component at low concentrations. We have used the method proposed by Colquhoun and Sakmann (1981) to analyze these brief closings and to extract estimates for the rates of channel opening (,3) and agonist dissociation (k-2). We find that this estimate of ,B does not predict our closed-time histograms at high agonist concentration (ACh: 30-300 ,uM; Carb: 300-1,000 uM). We conclude that brief closings at low agonist concentrations do not result solely from transitions between the doubly-liganded open and the doubly-liganded closed states. Instead, we postulate the existence of a second closed-channel state coupled to the open state.
The Nicotinic Acetylcholine Receptor, A Model of Ligand-Gated Ion Channels
The Jerusalem Symposia on Quantum Chemistry and Biochemistry, 1992
Acetylcholine (ACh) was one of the first neurotransmitters to be discovered and is one of the most important in the central nervous system (CNS) and peripheral nervous system (PNS). ACh is produced by the enzyme choline acetyltransferase and its actions are mediated through two types of acetylcholine receptors (AChRs) -the G protein-coupled muscarinic AChRs and the nicotinic AChRs (nAChRs).
The dissociation of acetylcholine from open nicotinic receptor channels
Proceedings of the National Academy of Sciences, 2001
Ligand-gated ion channels bind agonists with higher affinity in the open than in the closed state. The kinetic basis of this increased affinity has remained unknown, because even though the rate constants of agonist association to and dissociation from closed receptors can be estimated with reasonable certainty, the kinetics of the binding steps in open receptors have proven to be elusive. To be able to measure the agonist-dissociation rate constant from open muscle nicotinic receptors, we increased the probability of ligand unbinding from the open state by engineering a number of mutations that speed up opening and slow down closing but leave the ligand-binding properties unchanged. Single-channel patchclamp recordings from the wild-type and mutant constructs were performed at very low concentrations of acetylcholine (ACh). The durations of individual channel activations were analyzed assuming that ''bursts'' of fully liganded (diliganded) receptor openings can be terminated by ligand dissociation from the closed or open state (followed by fast closure) or by desensitization. This analysis revealed that ACh dissociates from diliganded open receptors at Ϸ24 s ؊1 , that is, Ϸ2,500 times more slowly than from diliganded closed receptors. This change alone without a concomitant change in the association rate constant to the open state quantitatively accounts for the increased equilibrium affinity of the open channel for ACh. Also, the results predict that both desensitization and ACh dissociation from the open state frequently terminate bursts of openings in naturally occurring gain-of-function mutants (which cause slow-channel congenital myasthenia) and therefore would contribute significantly to the time course of the endplate current decay in these disease conditions.
Glycine Hinges with Opposing Roles at the Acetylcholine Receptor-Channel Transmitter Binding Site
Biophysical Journal, 2011
The extent to which agonists activate synaptic receptor-channels depends on both the intrinsic tendency of the unliganded receptor to open and the amount of agonist binding energy realized in the channel-opening process. We examined mutations of the nicotinic acetylcholine receptor transmitter binding site (␣ subunit loop B) with regard to both of these parameters. ␣Gly147 is an "activation" hinge where backbone flexibility maintains high values for intrinsic gating, the affinity of the resting conformation for agonists and net ligand binding energy. ␣Gly153 is a "deactivation" hinge that maintains low values for these parameters. ␣Trp149 (between these two glycines) serves mainly to provide ligand binding energy for gating. We propose that a concerted motion of the two glycine hinges (plus other structural elements at the binding site) positions ␣Trp149 so that it provides physiologically optimal binding and gating function at the nerve-muscle synapse.
The effective opening of nicotinic acetylcholine receptors with single agonist binding sites
2011
Modern understanding of synaptic ion channels began with the isolation (Karlin and Cowburn, 1973) and subsequent cloning of nicotinic acetylcholine (ACh) receptors (nAChRs) (Numa et al., 1983). Based on the presence of primary binding site elements, including a pair of vicinal cysteines, 10 different nAChR subunits have been identified in vertebrates as subunits (1-10). Non- subunits, which demonstrably contain the required elements for forming the complementary surface of an agonist binding site, are , , and in muscle-type receptors and 2 and 4 in neuronal receptors. The nAChR ligand binding domain is formed by the interface of two protein subunits; the primary surface is formed by an subunit, which contains several other key elements in addition to the adjacent cysteines of a subdomain identified as the C-loop (Sine, 2002). The distinction between and non- subunits relates to a key dichotomy in several of the subfamilies of Cys-loop receptors between types that function as pentamers of an identical subunit (homomeric receptors) and those that require both and non- subunits in each penta-Correspondence to Roger L.
2010
Aromatic-aromatic interactions are a prominent feature of the crystal structure of ELIC [Protein Data Bank (PDB) code 2VL0], a bacterial member of the nicotinic receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow iris that occludes the transmembrane pore. To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine (ACh) receptor (one position at a time), including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays. From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines, rather than the accumulation of their independent effects, leads to the formation of a nonconductive conformation that is unresponsive to the application of ACh and is highly stable even in the absence of ligand. Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state. It seems reasonable to propose then that it is in this peculiar nonconductive conformation that the ELIC channel was crystallized. It seems also reasonable to propose that, in the absence of rings of porefacing aromatic side chains, such stable conformation may never be attained by the ACh receptor. Incidentally, we also noticed that the response of the proton-gated wild-type GLIC channel to a fast change in pH from pH 7.4 to pH 4.5 (on the extracellular side) is only transient, with the evoked current fading completely in a matter of seconds. This raises the possibility that the crystal structures of GLIC obtained at pH 4.0 (PDB code 3EHZ) and pH 4.6 (PDB code 3EAM) correspond to the to the (well-known) desensitized state.
ACS Chemical Biology, 2012
The nicotinic acetylcholine receptors (nAChRs) are a family of closely related but pharmacologically distinct neurotransmitter-gated ion channels. They are therapeutic targets for a wide range of neurological disorders, and a key issue in drug development is selective targeting among the greater than 20 subtypes of nAChRs that are known. The present work evaluates a proposed hydrogen bonding interaction involving a residue known as the "loop B glycine" that distinguishes receptors that are highly responsive to ACh and nicotine from those that are much less so. We have performed structure-function studies on the loop B site, including unnatural amino acid mutagenesis, in three different nAChR subtypes and found that the correlation between agonist potency and this residue is strong. Low potency receptor subtypes have a glycine at this key site, and mutation to a residue with a side chain converts a low potency receptor to a high potency receptor. Innately high potency receptors have a lysine at the loop B site and show a decrease in potency for the reverse mutation (i.e. introducing a glycine). This residue lies outside of the agonist binding site, and studies of other residues at the agonist binding site show that the details of how changes at the loop B glycine site impact agonist potency vary for differing receptor subtypes. This suggests a model in which the loop B residue influences the global shape of the agonist binding site rather than modulating any specific interaction.
Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins
Neuron, 1995
The nicotinic acetylcholine (ACh) receptors and the other neurotransmitter-gated ion channels have key roles in fast synaptic transmission throughout the nervous system. These receptors have a simple functional repertoire: they bind a specific neurotransmitter, open a gate, conduct specific ions across the membrane, and desensitize. Although it is easy for us to imagine in a general way how they might carry out these steps, to determine the actual mechanism requires more detailed structural information than we now have. Nevertheless, new information about the parts of these receptors that are in the front line of function, the neurotransmitter binding sites, the ion-conducting channel, and the gate, provides intriguing clues, albeit not always consistent ones, about the mechanisms of these receptors. Most progress toward understanding function in terms of structure has been made with the ACh receptors, on which we will focus, at the same time noting what is conserved and what is variable among all of the neurotransmitter-gated ion channels. The ACh receptors are members of a family of neurotransmitter-gated ion channels, which also includes receptors for y-aminobutyric acid (GABA), glycine (Gly), and 5hydroxytryptamine (5HT; Nodaet al., 1983; Grenningloh et al., 1987; Schofieldet al., 1987; Maricqet al., 1991); also in this family is an invertebrate glutamate-gated chloride channel (Gully et al., 1994). The subunits of these receptors have similar sequences and distributions of hydrophobic, membrane-spanning segments and are homologous (Figures 1 and 2). In this family, each subunit contains, in its N-terminal extracellular half, 2 cysteine (Cys) residues separated by 13 other residues. These Cys residues are disulfide linked in the ACh receptor (Kao and Karlin, 1986) and presumably in the homologous receptors, thereby closing a 15-residue loop. Because of this unique, invariant feature, we will call this family the Cys-loop receptors. The subunits of other ligand-gated ion channels-the receptors for glutamate (cation-conducting), for ATP, and for the second messengers, CAMP and cGMP-have sequences and distributions of putative membrane-spanning segments that are dissimilar from those of the Cysloop receptors (Figures 1 and 2). Despite the differences in their structures, all of these ligand-gated ion channels carry out the same general functions. This implies that, at the level of detailed mechanisms, there are many ways of recognizing specific ligands, of transducing binding into propagated structural changes, of gating a channel, and of selecting and conducting specific ions through a membrane. It is likely, however, that among the homologous Cys-loop receptors the mechanisms are very similar and that insight into one is applicable to all.