Novel Photoactivatable Agonist of the Nicotinic Acetylcholine Receptor of Potential Use for Exploring the Functional Activated State

Abstract: The nicotinic acetylcholine receptor (AChR) exhibits at least four different conformational states varying in affinity for agonists such as acetylcholine (ACh). Photoaffinity labeling has been previously used to elucidate the topography of the AChR. However, to date, the photosensitive probes used to explore the cholinergic binding site photolabeled only closed or desensitized states of the receptor. To identify the structural modifications occurring at the ACh binding site on allosteric transition associated with receptor activation, we have investigated novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as putative cholinergic agonists. Such compounds are fairly stable in the dark and generate highly reactive carbenic species on irradiation. In binding experiments using AChRs from Torpedo marmorata, these ligands had affinities for the ACh binding site in the micromolar range and did not interact with the noncompetitive blocker site (greater than millimolar affinity). Irreversible photoinactivation of ACh binding sites was obtained with the ligand 1b (up to 42% at 500 µM) in a protectable manner. In patch-clamp studies, 1b was shown to be a functional agonist of peripheral AChR in TE 671 cells, with the interesting property of exhibiting no or very little desensitization even at high concentrations.     

Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ～12 ? from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.     

Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.     

Acetylcholine Receptor Channels Activated by a Single Agonist Molecule

The neuromuscular acetylcholine receptor (AChR) is an allosteric protein that alternatively adopts inactive versus active conformations (R↔R^∗). The R^∗ shape has a higher agonist affinity and ionic conductance than R. To understand how agonists trigger this gating isomerization, we examined single-channel currents from adult mouse muscle AChRs that isomerize normally without agonists but have only a single site able to use agonist binding energy to motivate gating. We estimated the monoliganded gating equilibrium constant E₁ and the energy change associated with the R versus R^∗ change in affinity for agonists. AChRs with only one operational binding site gave rise to a single population of currents, indicating that the two transmitter binding sites have approximately the same affinity for the transmitter ACh. The results indicated that E₁ ≈ 4.3 × 10⁻³ with ACh, and ≈1.7 × 10⁻⁴ with the partial-agonist choline. From these values and the diliganded gating equilibrium constants, we estimate that the unliganded AChR gating constant is E₀ ≈ 6.5 × 10⁻⁷. Gating changes the stability of the ligand-protein complex by ∼5.2 kcal/mol for ACh and ∼3.3 kcal/mol for choline.     

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs.     

Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation

Acetylcholine (ACh) has been shown to modulate neuronal differentiation during early development. Both muscarinic and nicotinic acetylcholine receptors (AChRs) regulate a wide variety of physiological responses, including apoptosis, cellular proliferation and neuronal differentiation. However, the intracellular mechanisms underlying these effects of AChR signaling are not fully understood. It is known that activation of AChRs increase cellular proliferation and neurogenesis and that regulation of intracellular calcium through AChRs may underlie the many functions of ACh. Intriguingly, activation of diverse signaling molecules such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src is modulated by AChRs. Here we discuss the roles of ACh in neuronal differentiation, cell proliferation and apoptosis. We also discuss the pathways involved in these processes, as well as the effects of novel endogenous AChRs agonists and strategies to enhance neuronal-differentiation of stem and neural progenitor cells. Further understanding of the intracellular mechanisms underlying AChR signaling may provide insights for novel therapeutic strategies, as abnormal AChR activity is present in many diseases.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i     

Activation kinetics of recombinant mouse nicotinic acetylcholine receptors: mutations of alpha-subunit tyrosine 190 affect both binding and gating.

Affinity labeling and mutagenesis studies have demonstrated that the conserved tyrosine Y190 of the acetylcholine receptor (AChR) alpha-subunit is a key determinant of the agonist binding site. Here we describe the binding and gating kinetics of embryonic mouse AChRs with mutations at Y190. In Y190F the dissociation constant for ACh binding to closed channels was reduced approximately 35-fold at the first binding site and only approximately 2-fold at the second site. At both binding sites the association and dissociation rate constants were decreased by the mutation. Compared with wildtype AChRs, doubly-liganded alpha Y190F receptors open 400 times more slowly but close only 2 times more rapidly. Considering the overall activation reaction (vacant-closed to fully occupied-open), there is an increase of approximately 6.4 kcal/mol caused by the Y-to-F mutation, of which at least 2.1 and 0.3 kcal/mol comes from altered agonist binding to the first and second binding sites, respectively. The closing rate constant of alpha Y190F receptors was the same with ACh, carbamoylcholine, or tetramethylammonium as the agonist. This rate constant was approximately 3 times faster in ACh-activated S, W, and T mutants. The equilibrium dissociation constant for channel block by ACh was approximately 2-fold lower in alpha Y190F receptors compared with in wildtype receptors, suggesting that there are changes in the pore region of the receptor as a consequence of the mutation. The activation reaction is discussed with regard to energy provided by agonist-receptor binding contacts, and by the intrinsic folding energy of the receptor.     

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.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line

Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.     

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.     

Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

The relationship between the high-affinity procaine channel inhibition site (apparent dissociation constant Kp congruent to 200 microM) and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid 86Rb+ quenched-flux assays at 4 degrees C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with alpha-bungarotoxin (alpha-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations (10 microM-10 mM) alone, AChR undergoes one phase of inactivation (fast desensitization, rate = kd) in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting (greater than 10 mM) ACh concentrations [Forman & Miller (1988) Biophys. J. 54, 149-158]--rapid inactivation (rate = kr) complete in 30-75 ms is followed by fast desensitization at the same kd observed without procaine. The dependence of kr on [procaine] is consistent with a bimolecular association between procaine and its AChR site with kon = 2.5 X 10(5) M-1 s-1, koff = 36 s-1, and Kp = 145 +/- 36 microM). Inhibition of AChR function by mixtures of procaine (up to 12Kp) plus self-inhibiting concentrations of ACh or suberyldicholine ([SubCh] up to 13 X the 50% self-inhibiting agonist concentration, KB) was studied by reducing the level of alpha-BTX block in vesicles. The apparent KB increased in the presence of procaine, and the apparent KP increased linearly with [SubCh], indicating mutually exclusive actions at a common AChR site. Our data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and kr's dependence on [ACh] in the channel-activating range closely parallels that of 86Rb+ flux response to ACh.     

Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor.

The acetylcholine receptor (AChR) from vertebrate skeletal muscle is a pentamer composed of two ligand-binding alpha-subunits and one beta-, gamma-, and delta-subunit. To examine the functional roles of the non-alpha-subunits, we have expressed, in stable cell lines, AChRs lacking either a gamma- or a delta-subunit. Most previous work has examined how these changes in subunit composition affect single channel properties. Here, we take advantage of the stable expression system to produce large amounts of AChR and, for the first time, examine ligand binding to altered AChRs on intact cells. The changes in subunit composition affect both ligand affinity and cooperativity of the receptor, suggesting important roles for the gamma- and delta-subunits, both in shaping the ligand binding site and maintaining cooperative interactions between alpha-subunits.     

Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle

We used specific antibodies to gamma, delta, and epsilon subunits to characterize acetylcholine receptor (AChR) in extracts and at endplates of developing, adult, and denervated rat muscle. The AChRs in normal adult muscle were immunoprecipitated by anti-epsilon and anti-delta, but not by anti-gamma antibodies, whereas AChRs in denervated and embryonic muscles were precipitated by anti-gamma and anti-delta, but showed little or no reactivity to anti-epsilon antibodies. In immunofluorescence experiments, AChRs at neonatal endplates bound antibodies to gamma or delta, but not epsilon, subunit, whereas those in adult muscles bound antibodies to epsilon or delta, but not gamma, subunit. AChRs at denervated endplates and at developing endplates between postnatal days 9 and 16 bound all three antibodies. We conclude that the distribution of gamma and epsilon subunits of the AChR parallels the distribution of AChRs with embryonic and adult channel properties, respectively.     

Nicotinic acetylcholine receptors in health and disease

Nicotinic acetylcholine receptors (AChRs) are a family of acetylcholine-gated cation channels that form the predominant excitatory neurotransmitter receptors on muscles and nerves in the peripheral nervous system. AChRs are also expressed on neurons in lower amounts throughout the central nervous system. AChRs are even being reported on unexpected cell types such as keratinocytes. Structures of these AChRs are being determined with increasing precision, but functions of some orphan subunits are just beginning to be established. Functional roles for postsynaptic AChRs in muscle are well known, but in neurons the post-, peri-, extra-, and presynaptic roles of AChRs are just being revealed. Pathogenic roles of AChRs are being discovered in many diseases involving mechanisms ranging from mutations, to autoimmune responses, to the unknown; involving cell types ranging from muscles, to neurons, to keratinocytes; and involving signs and symptoms ranging from muscle weakness to epilepsy, to neurodegenerative disease, to psychiatric disease, to nicotine addiction. Awareness of AChR involvement in some of these diseases has provoked new interests in development of therapeutic agonists for specific AChR subtypes and the use of expressed cloned AChR subunits as possible immunotherapeutic agents. Highlights of recent developments in these areas will be briefly reviewed.     

Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.     

L-type calcium channels mediate acetylcholine receptor aggregation on cultured muscle

Agrin activation of muscle specific kinase (MuSK) initiates postsynaptic development on skeletal muscle that includes the aggregation of acetylcholine receptors (AChRs; Glass et al. [1996]: Cell 85: 513-523; Gautam et al. [1996]: Cell 85: 525-535). Although the agrin/MuSK signaling pathway remains largely unknown, changes in intracellular calcium levels are required for agrin-induced AChR aggregation (Megeath and Fallon [1998]: J Neurosci 18: 672-678). Here, we show that L-type calcium channels (L-CaChs) are required for full agrin-induced aggregation of AChRs and sufficient to induce agrin-independent AChR aggregation. Blockade of L-CaChs in muscle cultures inhibited agrin-induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Activation of L-CaChs in the absence of agrin induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Agrin responsiveness was significantly reduced in primary muscle cultures from the muscular dysgenesis mouse, a natural mutant, which does not express the L-CaCh. Our results establish a novel role for L-CaChs as important sources of the intracellular calcium necessary for the aggregation of AChRs.     

Characterization of an adult muscle acetylcholine receptor subunit by expression in fibroblasts.

To study the functional and structural roles of the epsilon subunit in adult muscle acetylcholine receptor (AChR), we have co-expressed the alpha and epsilon subunits of the mouse receptor in transfected fibroblasts. Ligand binding studies suggest that association of epsilon with alpha subunit results in a lower association rate constant for 125I-labeled alpha-bungarotoxin binding than that of the unassembled alpha subunit, approaching that for toxin binding to the AChR. Furthermore, alpha epsilon complexes contain high affinity binding sites for competitive antagonists and agonists not present in the unassembled alpha subunit, but similar to one of the two nonequivalent binding sites in the adult AChR. Structural analysis of alpha epsilon complexes by sucrose gradient velocity centrifugation suggests that some of the complexes formed are trimers or tetramers of alpha and epsilon subunits. Comparison of these data with those previously obtained for alpha gamma complexes suggests that gamma and epsilon have homologous functional roles and identical structural positions in the fetal and adult AChRs, respectively.     

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.