Mouse Muscle Acetylcholine Receptor Research Articles

Modal Gating of Muscle AChRs having Loop C Mutations

Many ion channels exhibit multiple patterns of kinetic activity (‘modes’) in single-channel currents. This behavior is rare in WT mouse muscle acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. We have found that mutations of all loop C residues at the transmitter binding site (except αY190 and αY198) increase the probability of modes. The free energy of diliganded gating is the sum of that for unliganded gating and from agonist affinity-changes at 2 binding sites: ΔG2=ΔG0+2ΔGB. For WT adult AChRs these values are (−100 mV; kcal/mol): −1.9, +8.3 and −5.1 (ACh). All mutations of αP197 (ACDEGIKSVY) had at least 2 gating modes with all agonists (ACh, Cho, CCh and TMA). We focused on αP197A and ACh and Cho. There were 3 distinct modes at saturating [agonist] but only 1 mode without agonist. Hence, the kinetic heterogeneity for this construct is generated by fluctuations in ΔGB. ΔΔGB was estimated for each mode (kcal/mol) for ACh (−1.7, WT and +1.6) and Cho (−2.2, WT and +0.9). That is, the gating equilibrium constant for one mode was ∼30-times larger than the WT (for both agonists), for another was about the same as the WT and for the third was ∼8-times smaller. An added F mutation at αY190 or αW149 reduced modal gating whereas one at αY198 or αY93 did not. An F substitution at eW55 had no effect, but one at δW57 reduced the modes. We also examined AChRs having only 1 active binding site and observed less heterogeneity from αP197A at the fetal α-γ site vs. the adult α-e or α-δ sites. Our working hypothesis is that mutations in loop C allow the agonist to sample multiple, meta-stable positions within the binding pocket, to produce distinct ΔGB values.

Linear Free Energy Relationships for Neurotransmitter Binding to a Nicotinic Acetylcholine Receptor

Agonists turn on receptors because they bind with a higher affinity to the active vs. resting form of their target sites. We measured the correlation between low-affinity (LA) and high-affinity (HA) neurotransmitter binding energies in adult mouse muscle acetylcholine receptors (AChRs) by using single-channel electrophysiology. The energy values were estimated from current interval durations, for different agonists and for mutations of seven different binding site amino acids. The results were analyzed as a linear free energy relationship, the slope of which gives the fraction of the overall binding chemical potential at the point the LA complex is established. All mutations of four binding site alpha-subunit aromatic amino acids (Y93, W149, Y190 and Y198) and alphaG147 produced fold-decreases in LA binding (relative to ACh, wild-type) that were about half those at in HA binding. For most residues (and agonists) this slope was ∼0.5, but mutations of two binding site residues had a significantly higher (0.90; alphaG153) or lower (0.25; epsilon P121) slope. The sequence of energy changes in the binding process appears to be alphaG153>(aromatics and alphaG147)>epsilonP121.

Effects of static magnetic fields on nicotinic cholinergic receptor function

Ligand-gated ion channel kinetics were studied in mammalian transfected cells encoding adult mouse muscle acetylcholine (ACh) receptors. We measured macroscopic and single-channel currents using the outside-out and cell-attached patch-clamp configurations. Cultured cells were exposed to moderate intensity inhomogeneous static magnetic fields up to 180 mT and measurements were performed for temperatures ranging from 5 to 50 °C. We found no significant changes in ACh-elicited macroscopic or single-channel currents. We observed the expected dependence in current decay constants with temperature, but negligible magnetic field influence on the channel's kinetics.

Mechanism of verapamil action on wild‐type and slow‐channel mutant human muscle acetylcholine receptor

Verapamil, a Ca(2+) channel blocker widely used in clinical practice, also affects the properties of frog and mouse muscle acetylcholine receptor (AChR). Here, we examine the mechanism of action of verapamil on human wild-type and slow-channel mutant muscle AChRs harboring in any subunit a valine-to-alanine mutation of 13' residue of the pore-lining M2 transmembrane segment. Verapamil, after a pre-treatment of 0.5-10 s, accelerated the decay of whole-cell or macroscopic outside-out currents within milliseconds of ACh application even at clinically attainable doses. Recordings of unitary events in the cell-attached and outside-out configurations showed that verapamil does not alter single-channel conductance, but reduces channel open probability, by prolonging the dwell time into the closed state for wild-type and all mutant AChR. The duration of channel openings decreased only for the epsilonV265A-AChR, by shortening the longest exponential component of the open-time distribution. These results provide a rationale for the therapeutic use of verapamil in the slow-channel syndrome and emphasize the major role played by epsilon subunit in controlling the functional properties of human muscle AChR, as revealed by the peculiar alterations imparted by mutations in this subunit.

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.

Activation and Inhibition of Mouse Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric alpha7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-beta-erythroidine as selective antagonists in mouse models of alpha3beta4 and alpha4beta2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal alpha and beta subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse alpha5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse alpha4beta2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity.

Is the inhibition of nicotinic acetylcholine receptors by bupropion involved in its clinical actions?

In this mini review we will focus on those molecular and cellular mechanisms exerted by bupropion (BP), ultimately leading to the antidepressant and anti-nicotinic properties described for this molecule. The main pharmacological mechanism is based on the fact that BP induces the release as well as inhibits the reuptake of neurotransmitters such as a dopamine (DA) and norepinephrine (NE). Additional mechanisms of action have been also determined. For example, BP is a noncompetitive antagonist (NCA) of several nicotinic acetylcholine receptors (AChRs). Based on this evidence, the dual antidepressant and anti-nicotinic activity of BP is currently considered to be mediated by its stimulatory action on the DA and NE systems as well as its inhibitory action on AChRs. Considering the results obtained in the archetypical mouse muscle AChR, a sequential mechanism can be hypothesized to explain the inhibitory action of BP on neuronal AChRs: (1) BP first binds to AChRs in the resting state, decreasing the probability of ion channel opening, (2) the remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process, and (3), BP interacts with a binding domain located between the serine (position 6′) and valine (position 13′) rings that is shared with the NCA phencyclidine and other tricyclic antidepressants. This new evidence paves the way for further investigations using AChRs as targets for the action of safer antidepressants and novel anti-addictive compounds.

Reverse transcription-polymerase chain reaction on a microarray: the integrating concept of “active arrays”

In this report we describe the proof of principle of a reverse transcription polymerase chain reaction (RT-PCR) but on-chip, with immobilized specific primers using a transcriptome of mouse-muscle fibroblasts for detection of muscle-specific expression products of these cells. The isolated total mRNA was directly incubated on an array of immobilized and solubilized specific primers, which allow the amplification of certain muscle-specific RNAs via its immobilized cDNAs. In contrast to others, the immobilized cDNA-products were directly synthesized on the chip by applying covalently bound specific primers. The products were detected by the incorporated and fluorophore-modified specific primers of the subsequently synthezised second strand. In addition, this second-strand served as a further template (like the basically used mRNA) in the subsequent solid-phase-PCR to amplify first-strand cDNA copies at the remaining immobilized specific primer-probes. This is the intrinsic factor of the amplification of certain signals of this application. The specific cDNA templates of genes coding for subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm), and ATPase (Atp2a2) were amplified on a biochip by RT-PCR directly from freshly isolated mRNA. The resulting procedure allows the detection of mRNA sequences from less than 5 pg of total RNA preparations.

Two Potent α3/5 Conotoxins from Piscivorous <italic>Conus achatinus</italic>

Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. alpha3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of alpha3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac1.1a and Ac1.1b, with appropriate disulfide bonding, based on cDNA sequences of alpha3/5 conotoxins from C. achatinus. Ac1.1a and Ac1.1b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC50 values of 36 nM and 26 nM, respectively. For Ac1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the N-terminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the alpha1-delta subunit interface instead of the alpha1-gamma binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor.

Roles of Amino Acids and Subunits in Determining the Inhibition of Nicotinic Acetylcholine Receptors by Competitive Antagonists

Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the alpha-epsilon interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. HEK293 cells were transfected with wild-type or mutant (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. Effects of mutations were antagonist specific. alphaY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates. Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.

The role of the amino acid residue at α1:189 in the binding of neuromuscular blocking agents to mouse and human muscle nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (AChRs) are valuable therapeutic targets. To exploit them fully requires rapid assays for the evaluation of potentially therapeutic ligands and improved understanding of the interaction of such ligands with their receptor binding sites. A variety of neuromuscular blocking agents (NMBAs) were tested for their ability to inhibit the binding of [(125)I]alpha-bungarotoxin to TE671 cells expressing human muscle AChRs. Association and dissociation rate constants for vecuronium inhibition of functional agonist responses were then estimated by electrophysiological studies on mouse muscle AChRs expressed in Xenopus oocytes containing either wild type or mutant alpha1 subunits. The TE671 inhibition binding assay allowed for the rapid detection of competitive nicotinic AChR ligands and the relative IC(50) results obtained for NMBAs agreed well with clinical data. Electrophysiological studies revealed that acetylcholine EC(50) values of muscle AChRs were not substantially altered by non-conservative mutagenesis of phenylalanine at alpha1:189 and proline at alpha1:194 to serine. However the alpha1:Phe189Ser mutation did result in a 3-4 fold increase in the rate of dissociation of vecuronium from mouse muscle AChRs. The TE671 binding assay is a useful tool for the evaluation of potential therapeutic agents. The alpha1:Phe189Ser substitution, but not alpha1:Pro194Ser, significantly increases the rate of dissociation of vecuronium from mouse muscle AChRs. In contrast, these non-conservative mutations had little effect on EC(50) values. This suggests that the AChR agonist binding site has a robust functional architecture, possibly as a result of evolutionary 'reinforcement'.

Gene Expression in the Brain from Fluoxetine‐Injected Mouse Using DNA Microarray

Previously we have examined the effects of phencyclidine and clozapine upon the gene expression in the mouse brain. Recently, fluoxetine (Prozac) has been introduced for the therapeutic purpose as an antidepressant drug. Miledi et al. reported blockage of mouse muscle and neuronal nicotinic acetylcholine receptor by various concentrations of fluoxetine. Furthermore, Kobayashi et al. discovered that fluoxetine inhibits G protein activated inwardly rectifying G protein activated K(+) (GIRK) channels using Xenopus oocyte expression assay. From these experiments, we considered that it might be interesting to study the effects of fluoxetine on the gene expression in the mouse brain. After we have injected fluoxetine once a day into mouse for 20 days, we sacrificed mouse by decapitation and extracted RNA from mouse cerebral cortex. We used DNA microarray method for examining the gene expression in the brain. We found the downregulation of many spot signals in the fluoxetine-treated mouse, for example cholecystockinin and prostaglandin D2 synthase.

Site-specific Charge Interactions of α-Conotoxin MI with the Nicotinic Acetylcholine Receptor

We have tested the importance of charge interactions for alpha-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR). Ionic residues on alpha-conotoxin MI were altered by site-directed mutagenesis or by chemical modification. In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2-4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR. It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium. Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the alphadelta site but not at the alphagamma site. It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the alphadelta site, and the two sites have distinct arrangements of charged residues. Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium. Thus, alpha-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.

Kinetic, Mechanistic, and Structural Aspects of Unliganded Gating of Acetylcholine Receptor Channels

The spontaneous activity of adult mouse muscle acetylcholine receptor channels, transiently expressed in HEK-293 cells, was studied with the patch-clamp technique. To increase the frequency of unliganded openings, mutations at the 12' position of the second transmembrane segment were engineered. Our results indicate that: (a) in both wild type and mutants, a C <--> O kinetic scheme provides a good description of spontaneous gating. In the case of some mutant constructs, however, additional states were needed to improve the fit to the data. Similar additional states were also needed in one of six patches containing wild-type acetylcholine receptor channels; (b) the delta12' residue makes a more pronounced contribution to unliganded gating than the homologous residues of the alpha, beta, and straightepsilon subunits; (c) combinations of second transmembrane segment 12' mutations in the four different subunits appear to have cumulative effects; (d) the volume of the side chain at delta12' is relevant because residues larger than the wild-type Ser increase spontaneous gating; (e) the voltage dependence of the unliganded gating equilibrium constant is the same as that of diliganded gating, but the voltage dependences of the opening and closing rate constants are opposite (this indicates that the reaction pathway connecting the closed and open states of the receptor changes upon ligation); (f) engineering binding-site mutations that decrease diliganded gating (alphaY93F, alphaY190W, and alphaD200N) reduces spontaneous activity as well (this suggests that even in the absence of ligand the opening of the channel is accompanied by a conformational change at the binding sites); and (g) the diliganded gating equilibrium constant is also increased by the 12' mutations. Such increase is independent of the particular ligand used as the agonist, which suggests that these mutations affect mostly the isomerization step, having little, if any, effect on the ligand-affinity ratio.

Altered Glycosylation Sites of the δ Subunit of the Acetylcholine Receptor (AChR) Reduce αδ Association and Receptor Assembly

We have used mutagenesis to investigate the potential N-glycosylation sites in the delta subunit of the mouse muscle acetylcholine receptor (AChR). Of the three sites, Asn76, Asn143, and Asn169, only the first two were glycosylated when the delta subunit was expressed in COS cells. Because the heterologously expressed delta subunit was similar in its properties to that expressed in C2 muscle cells, the sites of glycosylation are likely to be the same in both cases. In COS cells, mutations of the delta subunit that prevented glycosylation at either of the sites did not change its metabolic stability nor its steady-state level. These results are in contrast to those found previously for the alpha subunit, in which glycosylation at a single site metabolically stabilized the polypeptide (Blount, P., and Merlie, J. P. (1990) J. Cell Biol. 111, 2613-2622). Mutations of the delta subunit that prevented glycosylation, however, decreased its ability to form an alpha delta heterodimer when the alpha and delta subunit were expressed together. When all four subunits of the AChR (alpha, beta, delta, and epsilon) were coexpressed, mutation of the delta subunit to prevent glycosylation resulted in a reduced amount of fully assembled AChR and reduced surface AChR levels, consistent with the role of the heterodimer in the assembly reaction. These results suggest that glycosylation of the delta subunit at both Asn76 and Asn143 is needed for its efficient folding and/or its subsequent interaction with the alpha subunit.

Acetylcholine and Epibatidine Binding to Muscle Acetylcholine Receptors Distinguish between Concerted and Uncoupled Models

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.

5-hydroxytryptamine blocks the fetal more potently than the adult mouse muscle acetylcholine receptor.

The action of 5-hydroxytryptamine (5-HT) on fetal (gamma-AChR) and adult (epsilon-AChR) muscle acetylcholine receptors was studied in transfected BOSC 23 cells expressing mouse AChRs and in acutely dissociated mouse muscle fibres. In transfected cells, coapplication of 5-HT (0.01-10 mM) with ACh reversibly reduced the amplitude and accelerated the decay of the ACh-activated whole-cell currents, in a dose- and voltage-dependent manner. 5-HT blocked faster and with higher potency the gamma-AChR than the epsilon-AChR. Cell-attached recordings from transfected BOSC 23 cells and embryonic or neonatal muscle fibres were made. When 5-HT (5 microM) was included in the patch pipette, ACh-evoked unitary events acquired a bursting behaviour, which was more pronounced for gamma- than epsilon-AChR, though it was enhanced by membrane hyperpolarization for both AChR species. There was no effect on the single-channel conductance. It is concluded that 5-HT behaves as an open-channel blocker of muscle AChRs, with higher efficacy on gamma- than on epsilon-AChR.

Physicochemical and immunological studies of the N-terminal domain of the Torpedo acetylcholine receptor alpha-subunit expressed in Escherichia coli.

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of alpha2 beta gamma delta subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1-209 of the Torpedo californica AChR alpha-subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1-209 sequence, were used and the recombinant proteins obtained (designated alpha1-209pET and alpha1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]alpha-bungarotoxin with equal potency (KD approximately 130 nm, Bmax approximately 10 nmol.mg-1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly beta-structure (50-56%), the fraction of alpha-helices amounting to 32-35%. Nonionic (beta-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of beta-structure or increased the alpha-helicity, respectively. The predominance of beta-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR alpha-subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem. 272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR alpha-subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.

Nicotinic receptors are regulated by protein kinase C activated via a nicotinic receptors-mediated signaling pathway

The present study was conducted to examine the effect of protein kinase C (PKC) on nicotinic acetylcholine (ACh) receptors expressed in Xenopus oocytes by monitoring single-channel currents. In an outside-out patch-clamp configuration, ACh (1 μM) elicited single channel currents with a slope conductance of 31 pS (control) in normal Torpedo ACh receptors. Activation of PKC via an endogenous phosphatidylinositol signaling pathway elevated the slope conductance to 41 pS, which effect was blocked by the selective PKC inhibitor, staurosporine. Mutant ACh receptor channels, which mimic PKC phosphorylation of the receptors, exhibited a slope conductance of 41 pS. Notably, pretreatment with a higher concentration of ACh (100 μM) caused an increase in the slope conductance of the channels for 1 μM ACh (43 pS), which was the same level as obtained with either PKC activation or mutant ACh receptors, and this effect was also inhibited by staurosporine. In addition, the control slope conductance was reduced by PKC inhibitor peptide (24 pS), which corresponded to that obtained with another mutant ACh receptors lacking PKC phosphorylation sites (18 pS). Mouse muscle ACh receptors were also regulated by the same mechanism. The results of the present study suggest that ACh activates PKC via nicotinic ACh receptors, which alternatively, modulates the properties of the receptor channels.

Functional effects on the acetylcholine receptor of multiple mutations of gamma Asp174 and delta Asp180.

Residues gamma Asp174 and delta Asp180, previously implicated in the binding of acetylcholine (ACh) by the mouse muscle ACh receptor, were each mutated to nine other residues, Asn, Glu, Thr, Ala, Cys, His, Val, Tyr, and Lys. The effects of the mutations on ACh-induced current was determined on surface receptors containing wild-type alpha and beta subunits and mutant gamma and delta subunits. The mutations increased the concentration of ACh eliciting half-maximal current (EC50) by factors from 22 for the Glu mutant to 660 for the Lys mutant. Analysis of the effects in terms of the difference in the accessible surface areas of the mutant and wild-type side chains and the difference in side-chain charges indicated that, per binding site, Delta DeltaG0 for activation was a sum of 10 cal mol-1 A-2 of change in side-chain accessible surface area and of 0.95 kcal mol-1 positive step-1 in side-chain charge, equivalent to 1 mol of charge falling through 42 mV. The effects on the concentration of ACh (IC50, ACh) and of d-tubocurarine (IC50,dTC) causing half-maximal retardation of alpha-bungarotoxin binding were determined on complexes containing wild-type alpha and beta subunits and either mutant gamma or mutant delta subunit. The effects on IC50,ACh correlated well with the effects on EC50, with a similar magnitude for the influence of side-chain charge on the free energy of binding (in this case to the desensitized state) and on the electrostatic potential at the binding site. The effects on IC50,dTC were in all cases less than the effects on IC50,ACh, and the two sets of effects were poorly correlated. In line with the higher ACh affinity and lower d-tubocurarine affinity of the alpha-delta binding site compared to the alpha-gamma binding site, mutations of delta Asp180 had a greater effect on IC50,ACh than did the same mutations of gamma Asp174, and vice versa for effects on IC50,dTC. Consequently, all mutations decreased the asymmetry in the binding properties of the two types of sites.