Activation of the nicotinic acetylcholine receptor involves a switch in conformation of the alpha subunits

The nicotinic acetylcholine (ACh) receptor belongs to a superfamily of synaptic ion channels that open in response to the binding of chemical transmitters. Their mechanism of activation is not known in detail, but a time-resolved electron microscopic study of the muscle-type ACh receptor had suggested that a local disturbance in the ligand-binding region and consequent rotations in the ligand-binding alpha subunits, connecting to the transmembrane portion, are involved. A more precise interpretation of this structural change is given here, based on comparison of the extracellular domain of the ACh receptor with an ACh-binding protein (AChBP) to which a putative agonist is bound. We find that, to a good approximation, there are two alternative extended conformations of the ACh receptor subunits, one characteristic of either alpha subunit before activation, and the other characteristic of all three non-alpha subunits and the protomer of AChBP. Substitution in the three-dimensional maps of alpha by non-alpha subunits mimics the changes seen on activation, suggesting that the structures of the alpha subunits are modified initially by their interactions with neighbouring subunits and switch to the non-alpha form when ACh binds. This structural change, which entails 15-16 degrees rotations of the inner pore-facing parts of the alpha subunits, most likely acts as the trigger that opens the gate in the membrane-spanning pore.     

A model of the muscarinic receptor-induced changes in K(+)-current and action potentials in the bullfrog atrial cell.

A model is formulated for characterizing the behavior of the acetylcholine (ACh)-sensitive K+ membrane channel (muscarinic channel) in bullfrog atrial myocytes. Parameters of the muscarinic current model are chosen in fit available data from the literature on bullfrog atrial myocytes (3, 4, 45). This model is subsequently incorporated into a large mathematical model of the bullfrog myocyte that is based on quantitative whole-cell voltage clamp data (40). Simulations are conducted on the active atrial cell model in bathing media containing ACh at different concentrations to explore the effect of this muscarinic channel on the electrical behavior of the myocyte. The model predicts a progressive shortening of the action potential with increasing [ACh], as well as an indirect influence of the muscarinic K+ current on the other membrane currents of the atrial cell. Interpretation of the simulation results provides suggestions for the probable mechanisms underlying the shortening of the action potential due to activity of the muscarinic channel. Specifically, the model predicts that with an increase in ACh concentration: (a) the outward muscarinic current, IK,ACh(t), increases in magnitude but shortens in duration; (b) the calcium current, ICa(t), may increase in magnitude, but when it does so it decreases in duration compared with the control conditions; (c) the intracellular Ca2+ concentration [Ca2+]i waveform during the action potential decreases in both magnitude and duration. Because the contractile activity of the cell is controlled by the [Ca2+]i waveform, the model predicts a decrease in contractile strength with an increase in ACh concentration in the bathing medium; i.e., a negative inotropic effect.     

Some properties of acetylcholine receptors in human cultured myotubes

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [125I]alpha-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 +/- 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40-45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 microM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. -70 mV, 22 degrees C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 microM). With desensitizing doses of ACh (10 microM), channel openings occurred in obvious bursts; each burst usually appeared as part of a 'cluster' of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.     

Ligand-induced conformational change in the alpha7 nicotinic receptor ligand binding domain

Molecular dynamics simulations of a homology model of the ligand binding domain of the alpha7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca(2+), to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca(2+) appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change.     

Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G-proteins to activate ion channels.

Following the infection of insect ovarian cells (Sf9) with recombinant bearing the cDNA coding for the rat muscarinic acetylcholine (ACh) receptor subtype m3, ionic flux across the membrane in response to the application of ACh was examined electrophysiologically. We show that ACh activates potassium currents. The response is abolished when cells are treated with pertussis toxin. No ACh-induced currents are observed from uninfected cells or cells infected with virus which do not contain the cDNA coding for ACh receptors in its genome. The characteristics of single channel currents show time-dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 pS. The conductance level of channels gradually increases in steps to 10 pS and then to 20 pS and 40 pS. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synchrony with, the original channel.     

Bridging the Gap between Structural Models of Nicotinic Receptor Superfamily Ion Channels and Their Corresponding Functional States

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 pore-facing 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.     

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.     

Opening rate of acetylcholine receptor channels.

The nicotinic acetylcholine (ACh) receptor is responsible for rapid conversion of chemical signals to electrical signals at the neuromuscular junction. Because the receptor and its ion channel are components of a single transmembrane protein, the time between ACh binding and channel opening can be minimized. To determine just how quickly the channel opens, we made rapid (100-400 microseconds) applications of 0.1-10 mM ACh to outside-out, multichannel membrane patches from BC3H-1 cells, while measuring the onset of current flow through the channels at 11 degrees C. Onset time is steeply dependent upon ACh concentration when channel activation is limited by binding of ACh (0.1-1 mM). At +50 mV, the 20-80% onset time reaches a plateau near 110 microseconds above 5 mM ACh as channel opening becomes rate limiting. Thus, we calculate the opening rate, beta = 12/ms, without reference to specific channel activation schemes. At -50 mV, the combination of a rapid, voltage-dependent block of channels by ACh with a finite solution exchange time distorts onset. To determine opening rate at -50 mV, we determine the kinetic parameters of block from "steady-state" current and noise analyses, assume a sequential model of channel activation/block, and numerically simulate current responses to rapid perfusion of ACh. Using this approach, we find beta = 15/ms. In contrast to the channel closing rate, the opening rate is relatively insensitive to voltage.     

Competitive potentiation of acetylcholine effects on neuronal nicotinic receptors by acetylcholinesterase-inhibiting drugs

The effects of the acetylcholinesterase inhibitors physostigmine and tacrine on alpha4beta2 and alpha4beta4 subtypes of neuronal nicotinic acetylcholine (ACh) receptors, expressed in Xenopus laevis oocytes, have been investigated. In voltage-clamp experiments low concentrations of physostigmine and tacrine potentiate ion currents induced by low concentrations of ACh, whereas at high concentrations they inhibit ACh-induced ion currents. These dual effects result in bell-shaped concentration-effect curves. Physostigmine and tacrine, by themselves, do not act as nicotinic receptor againsts. The larger potentiation is observed with 10 microM: physostigmine on alpha4beta4 nicotinic receptors and amounts to 70% at 1 microM: ACh. The mechanism underlying the effects of physostigmine on alpha4beta4 ACh receptors has been investigated in detail. Potentiation of ACh-induced ion current by low concentrations of physostigmine is surmounted at elevated concentrations of ACh, indicating that this is a competitive effect. Conversely, inhibition of ACh-induced ion current by high concentrations of physostigmine is not surmounted at high concentrations of ACh, and this effect appears mainly due to noncompetitive, voltage-dependent ion channel block. Radioligand binding experiments demonstrating displacement of the nicotinic receptor agonist (125)I-epibatidine from its recognition sites on alpha4beta4 ACh receptors by physostigmine confirm that physostigmine is a competitive ligand at these receptors. A two-site equilibrium receptor occupation model, combined with noncompetitive ion channel block, accounts for the dual effects of physostigmine and tacrine on ACh-induced ion currents. It is concluded that these acetylcholinesterase-inhibiting drugs interact with the ACh recognition sites and are coagonists of ACh on alpha4-containing nicotinic ACh receptors.     

Effects of Ca2+ channel antagonists on acetylcholine and catecholamine releases in the in vivo rat adrenal medulla

To elucidate the types of voltage-dependent Ca(2+) channels controlling ACh and catecholamine releases in the in vivo adrenal medulla, we implanted microdialysis probes in the left adrenal medulla of anesthetized rats and investigated the effects of Ca(2+) channel antagonists on ACh, norepinephrine, and epinephrine releases induced by nerve stimulation. The dialysis probes were perfused with Ringer solution containing a cholinesterase inhibitor, neostigmine. The left splanchnic nerves were electrically stimulated at 2 and 4 Hz before and after intravenous administration of Ca(2+) channel antagonists. omega-Conotoxin GVIA (an N-type Ca(2+) channel antagonist, 10 microg/kg) inhibited ACh release at 2 and 4 Hz by approximately 40%, norepinephrine release at 4 Hz by approximately 50%, and epinephrine release at 2 and 4 Hz by approximately 45%. A fivefold higher dose of omega-conotoxin GVIA (50 microg/kg) did not further inhibit these releases. omega-Conotoxin MVIIC (a P/Q-type Ca(2+) channel antagonist, 50 microg/kg) inhibited ACh and epinephrine releases at 4 Hz by approximately 30%. Combined omega-conotoxin GVIA (50 microg/kg) and MVIIC (250 microg/kg) inhibited ACh release at 2 and 4 Hz by approximately 70% and norepinephrine and epinephrine releases at 2 and 4 Hz by approximately 80%. Nifedipine (an L-type Ca(2+) channel antagonist, 300 and 900 microg/kg) did not change ACh release at 2 and 4 Hz; however, nifedipine (300 microg/kg) inhibited epinephrine release at 4 Hz by 20%, and nifedipine (900 microg/kg) inhibited norepinephrine and epinephrine releases at 4 Hz by 30%. In conclusion, both N- and P/Q-type Ca(2+) channels control ACh release on preganglionic splanchnic nerve endings while L-type Ca(2+) channels do not. L-type Ca(2+) channels are involved in norepinephrine and epinephrine releases on chromaffin cells.     

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.     

A molecular scheme for the reaction between acetylcholine and nicotinic channels.

In outside-out patches of mouse-muscle membrane, embryonic-like channels were activated by pulses of acetylcholine (ACh). On increasing the ACh concentration, the rate of desensitization, 1/tau d, increased linearly with the peak open probability, indicating desensitization from the open state. Desensitization had only one time constant tau d at each ACh concentration. Recovery from desensitization was only approximately 10 times slower than desensitization, whereas the probability of steady-state channel opening, declined to < 0.01 with > 10(-6) M ACh. The peak probability of opening in > 10(-4) M ACh pulse was close to 1. A linear reaction scheme was not compatible with these results. The scheme had to be expanded resulting in a circular scheme with two additional ACh binding steps to desensitized channel states. The approximate rate constants of all reaction steps in the circular scheme could be determined using computer simulations. The model predicted that clusters of channel opening had the average duration tau d at the respective ACh concentration. In cell-attached patches on intact muscle fibers, similar average cluster durations were observed at the respective ACh concentration. This indicates that tau d in the intact muscle fibers has similar values as in outside-out patches.     

Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.     

Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall.

The nicotinic acetylcholine (ACh) receptor is the neurotransmitter-gated ion channel responsible for the rapid propagation of electrical signals between cells at the nerve/muscle synapse. We report here the 4.6 A structure of this channel in the closed conformation, determined by electron microscopy of tubular crystals of Torpedo postsynaptic membranes embedded in amorphous ice. The analysis was conducted on images recorded at 4 K with a 300 kV field emission source, by combining data from four helical families of tubes (-16,6; -18,6; -15,7; -17,5), and applying three-dimensional corrections for lattice distortions. The study extends earlier work on the same specimen at 9 A resolution.Several features having functional implications now appear with better definition. The gate of the channel forms a narrow bridge, consisting of no more than one or two rings of side-chains, across the middle portion of the membrane-spanning pore. Tunnels, framed by twisted beta-sheet strands, are resolved in the extracellular wall of the channel connecting the water-filled vestibule to the putative ACh-binding pockets. A set of narrow openings through which ions can flow are resolved between alpha-helical segments forming part of the cytoplasmic wall of the channel. It is suggested that the extracellular tunnels are access routes to the binding pockets for ACh, and that the cytoplasmic openings serve as filters to exclude anions and other impermeant species from the vicinity of the pore. Both transverse pathways are likely to be important in achieving a rapid postsynaptic response.     

Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities.

The experiments described examine single channel currents recorded through Torpedo acetylcholine receptor channels stably expressed by a mouse fibroblast cell line. Closed-duration histograms were constructed from currents elicited by 0.5-300 microM acetylcholine (ACh). The concentration dependence of closed durations is well described by a four-state linear scheme with the addition of open-channel block by ACh. Analysis of closed durations measured at low concentrations gives estimates of the rate of opening of doubly liganded receptors, beta, the rate of dissociation of ACh from doubly liganded receptors, k-2, and the rate of channel closing, alpha. The rate of ACh dissociation from singly liganded receptors, k-1, is then deduced from closed-duration histograms obtained at intermediate ACh concentrations. With k-1, k-2 and beta determined, the rates of ACh association, k+1 and k+2, are estimated from fitting closed-duration histograms obtained over a range of high ACh concentrations. A complete set of rate constants is presented for three experimental conditions: (a) Ca2(+)-free extracellular solution containing 1 mM free Mg2+ at 22 degrees C, (b) Ca2(+)-free solution at 12 degrees C, and (c) extracellular Ca2+ and Mg2+, both at 0.5 mM, at 22 degrees C. For all three conditions the dissociation constant for the first agonist binding site is approximately 100-fold lower than that for the second site. The different affinities are due primarily to different dissociation rates. Both the association and dissociation rates depend strongly on temperature. At 22 degrees C ACh associates at diffusion-limited rates, whereas at 12 degrees C association is 30- to 60-fold slower. Also slowed at 12 degrees C are beta (4-fold), k-2 (3-fold), k-1 (25-fold), and alpha (15-fold). In contrast to the activation rate constants, those for ACh-induced block decrease only twofold between 22 and 12 degrees C. Changing from a Ca2(+)-free to a Ca2(+)-containing extracellular solution does not affect k+1 and k+2, but increases beta (twofold) and decreases k-2, k-1, and alpha (all twofold). Spectral analysis of single channel currents supports the parameter estimates obtained from fitting the open- and closed-duration histograms, and improves resolution of brief channel blockages produced by ACh.     

The molecular structure of the receptor-ionophore complex at the neuromuscular junction.

A general theory of the molecular structure of receptors for transmitters based only on protein has been presented elsewhere (Smythies, 1974a,b). The acetylcholine receptor at the neuromuscular junction is postulated in particular to be based on a Kusnetsov-Ghokov grid with four sequencestwo “primary” chains A-x-B-cys-A-x-B where A = arg or lys and B = glu or phosphoser and two “secondary” chains of sequence -gly-x-gly-pro-x-ile-cys-asp-x- forming a symmetrical receptor cup of rectangular form. The present paper extends the model to include the gate over the adjacent ionophore (or “ion conductance modulator”: ICM) and the linking mechanism from receptor to gate. These are postulated to consist of a second Kusnetsov-Ghokov grid generated by a third “primary” chain along the side that covers the orifice to the ion conducting channel. The action of ACh is postulated to be to displace an hydrated Ca⁺⁺ ion from the receptor cup and to disrupt the AB rungs in the receptor grid. The middle primary chain then slides 14 Å and the AB links reform. This replaces a bulky amino acid pair normally blocking the ion channel by a less bulky amino acid pair and so hydrated ions can be transmitted. It is further postulated that snake neurotoxins (ACh blockers) in a specified conformation bind mainly to the ionophore grid and prevent the sliding filament mechanism from opening; whereas the snake “cardiotoxins” (ACh agonists)—in a specified conformation—bind to the same sliding filament mechanism in its “open” ionophore gate and prevent it being closed: and histrionicotoxin binds to the same open “gate” but blocks it physically. The hypothesis may rigorously be tested by experiment as it makes detailed predictions on the X-ray structure of the snake neurotoxins and cardiotoxins.     

Mapping the structural requirements for nicotinic acetylcholine receptor activation by using tethered alkyltrimethylammonium agonists and antagonists

A molecule as simple in structure as tetramethylammonium gates the nicotinic acetylcholine receptor (nAChR) with high efficacy. To compare the structure of the nAChR transmitter binding site in the open channel state with that of the ACh binding protein, we determined the efficacy of nAChR gating by -S(CH(2))(n)N(CH(3))(3)(+) (n = 1-4) tethered to substituted cysteines at positions in the alpha subunits or gamma and delta subunits predicted to contribute to the ACh binding sites in mutant Torpedo nAChRs expressed in Xenopus oocytes. For tethered thiocholine [-S(CH(2))(2)N(CH(3))(3)(+)], we previously reported that within alpha195-201 gating was observed only at alphaY198C while at alphaY93C it acted as an antagonist. We now show that within alpha191-194, thiocholine activates when tethered at alphaCys192 or alphaCys193. Thiocholine also activates when tethered at alphaY190C or alphaW149C in nAChRs containing a beta subunit mutation (betaL257S) that destabilizes the closed channel, but not from gammaW55C/deltaW57C, where longer adducts can activate. When tethered at positions in binding site segment E, thiocholine activates only from gammaL119C/deltaL121C, where the shorter -S(CH(2))(1)N(CH3)(3)(+) acts as an antagonist. Longer adducts tethered at gammaL109C/deltaL111C or gammaL119C/deltaL121C also activate, but less efficiently. The length requirements for efficient gating by tethered agonists agree closely with predictions based upon the structure of the agonist site in a nAChR homology model derived from the ACh binding protein structure, which suggests that this structure is an excellent model of the nAChR agonist binding site in the open channel conformation. The inability of thiocholine to activate from alphaY93C, which is not predicted by the model, is discussed in terms of the structure of the nAChR in the closed state.     

Inhibition of Na+,K+-ATPase by Ouabain Opens Calcium Channels Coupled to Acetylcholine Release in Guinea Pig Myenteric Plexus

Abstract: Ouabain, an Na⁺,K⁺-ATPase inhibitor, increases the release of acetylcholine (ACh) from various preparations in a Ca²⁺-independent way. However, in other preparations the release of ACh evoked by ouabain is dependent on the presence of extracellular calcium. In the present study, we have labeled the ACh of myenteric plexus longitudinal muscles of guinea pig ileum and compared the effect of calcium channel blockers on ouabain-evoked release of [³H]ACh. Release of [³H]ACh evoked by ouabain is dose dependent and decreased markedly in the absence of calcium or in the presence of cadmium, a nonspecific calcium channel blocker. N-type calcium channel blockage by the ω-conotoxins GVIA (selective N-type calcium channel blocker) and MVIIC (a nonselective calcium channel blocker) inhibited by 45 and 55%, respectively, the release of [³H]ACh. L-type calcium channel suppression by low concentrations of verapamil, nifedipine, and diltiazem had no effect on the release of [³H]ACh. The release of transmitter was also not affected significantly by nickel, a T-type calcium channel blocker. In addition, ω-agatoxin-IVA, at concentrations that block P- and Q-type calcium channels, did not affect significantly the release of [³H]ACh. Thus, extracellular Ca²⁺ is essential for the release of ACh induced by ouabain from guinea pig ileum myenteric plexus. In this preparation, the N-type calcium channel plays a dominant role in transmitter release evoked by inhibition of Na⁺,K⁺-ATPase, but other routes of calcium entry in addition to these channels can also support the release of neurotransmitter induced by ouabain.     

Analysis of the negative inotropic effect of acetylcholine on frog atrial fibres

Voltage-clamp experiments have been performed on frog atrial preparations in order to study the mechanism of the inotropic effect of acetylcholine (ACh) at various concentrations. The amplitude of the slow inward current (Is) is reduced even at low ACh concentrations; such low concentrations have little or no effect on potassium permeability. Dose-effect relationships for Is inhibition (Is/Is max) by ACh show a half amplitude dose (K0.5 around 8 X 10(-8) M ACh. The reduction of Is is attributed largely to a decrease of the maximal conductance of the slow channel (gs). Steady-state activation and inactivation parameters are not affected by ACh. Experiments in a Na-free solution (Na replaced by Li ions) or in a Ca-free solution (with EGTA) indicate that the "slow sodium current" is more sensitive to ACh than the "slow Ca current", although these two currents both seem to flow through the slow channel. The decrease of the phasic component of contraction observed in the presence of ACh is very well correlated with the decrease of Is (K0.5 = 8 X 10(-8) M ACh), while the increase of the tonic tension may be related to the outward potassium current induced by high concentrations of ACh. The significant difference between the half amplitude dose (K0.5) observed in the dose effect curves with ACh for Is inhibition (K0.5 = 8 X 10(-8) M) and for ACh-induced extra-current (K0.5 - 10(-6) M) may indicate the presence of two muscarinic receptors.     

Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor

We examined functional consequences of intrasubunit contacts in the nicotinic receptor alpha subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues alphaK145, alphaD200, and alphaY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations alphaD200N and alphaK145Q impair channel gating to the same extent as either single mutation, while alphaK145E counteracts the impaired gating due to alphaD200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, alphaK145 and alphaD200 form a salt bridge associated with the closed state of the channel. When ACh binds, alphaY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches alphaK145, which in turn loosens its contact with alphaD200. The positional changes of alphaK145 and alphaD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of beta-strand 7, which harbors alphaK145 and is part of the signature Cys-loop, and the second is of beta-strand 10, which harbors alphaD200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel.