Inhibition of nicotinic acetylcholine receptor by philanthotoxin-343: kinetic investigations in the microsecond time region using a laser-pulse photolysis technique.

The mechanism of inhibition of a nicotinic acetylcholine receptor (nAChR) in BC(3)H1 muscle cells by philanthotoxin-343 (PhTX-343), a synthetic analogue of philanthotoxin-433, a wasp toxin, was investigated using a laser-pulse photolysis technique with microsecond time resolution and in a carbamoylcholine concentration range of 20-100 microM and PhTX-343 concentration range of 0-200 microM. The rate constant for nAChR channel opening determined by the chemical kinetic techniques decreased with increasing PhTX-343 concentration, whereas there was no significant effect on the rate constant for channel closing. The resulting decrease in the channel-opening equilibrium constant accounted quantitatively for the inhibition of the receptor by the toxin. Single-channel current measurements suggest an additional step in which the open channel:inhibitor complex isomerizes to a nonconducting receptor form. Cell-flow experiments with a time resolution of 10 ms indicate that this isomerization step is only important at very high inhibitor concentrations. The inhibitor binds to the open-channel receptor form, with an affinity that is at least 5 times smaller than that for the closed-channel form. This indicates that receptor inhibition mainly involves the binding of PhTX-343 to the closed-channel form of the receptor. PhTX-343, and an analogue of this polyamine, had no effect when applied to the inside of the cell membrane. However, there was significant inhibition of the nAChR when these compounds were applied to the outside of the cell membrane, indicating an extracellular site for inhibition. Furthermore, increasing the transmembrane potential results in a decrease in the ability of PhTX-343 to inhibit the receptor. This observation is related to the voltage dependence of the effect of PhTX-343 on the rate constant for nAChR channel opening with increasing transmembrane voltage (-60 to 50 mV). This suggests that the voltage dependence of inhibition mainly reflects the effect of transmembrane voltage on the rate constant of channel opening and, therefore, the channel-opening equilibrium constant. PhTX-343 competes with cocaine and procaine for its binding site. The finding that this toxin, which binds to a common inhibitory site with compounds such as cocaine, exerts its effect by decreasing the channel-opening equilibrium constant suggests an approach for the development of therapeutic agents. A compound that binds to this regulatory site but does not affect the channel-opening equilibrium constant may be developed. Such a compound can displace an abused drug such as cocaine and thereby alleviate the toxic effect of this compound on the organism.     

Picrotoxin inhibition mechanism of a gamma-aminobutyric acid A receptor investigated by a laser-pulse photolysis technique

The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin. Here, we present the mechanism of inhibition by picrotoxin of the rat alpha1beta2gamma2L GABA(A) receptor investigated using rapid kinetic techniques in combination with whole-cell current recordings. The following new results were obtained by using transient kinetic techniques, the cell-flow method and the laser-pulse photolysis (LaPP) technique with a microsecond to millisecond time resolution. (i) The apparent dissociation constant of picrotoxin for the open-channel form of the receptor was approximately 5 times higher than that of the closed-channel form. (ii) Picrotoxin increased the channel-closing rate constant (k(cl)) approximately 4-fold, while the rate constant for channel opening (k(op)) remained essentially unaffected. (iii) The mechanism indicates that picrotoxin binds to an allosteric site of the receptor with higher affinity for the closed-channel form than for the open-channel form and thereby inhibits the receptor by decreasing 4-fold its channel-opening equilibrium constant [Phi(I)(-)(1) = k(op(I))/k(cl(I))]. (iv) The mechanism further indicates that compounds that bind with equal affinity to the picrotoxin-binding site on the open-channel form of the receptor and the closed-channel form will not affect the channel-opening equilibrium and can, therefore, displace picrotoxin and prevent inhibition of the GABA(A) receptor by picrotoxin. Such compounds may be therapeutically useful in counteracting the effects of compounds and diseases that unfavorably affect the channel-opening equilibrium of the receptor channel.     

Mechanism-based discovery of small molecules that prevent noncompetitive inhibition by cocaine and MK-801 mediated by two different sites on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) belongs to a group of five structurally related membrane proteins that play a major role in the communication between approximately 10(12) cells of the mammalian nervous system. The receptor is inhibited by both abused drugs and therapeutic agents. During the past two decades, many attempts have been made to find compounds that prevent cocaine inhibition of this protein. The use of newly developed transient kinetic techniques in investigations of the inhibition of the receptor by cocaine and MK-801 led to an inhibition mechanism not previously proposed. It was observed that the receptor contains two inhibitory sites: one that equilibrates with the tested noncompetitive inhibitors within approximately 50 ms, and a second site that equilibrates with inhibitors within approximately 1 s. The mechanism of inhibition of the rapidly equilibrating inhibitory site has been investigated, and based on that mechanism, the first evidence that small organic molecules exist that prevent inhibition of the rapidly equilibrating inhibitory site was obtained. These compounds did not prevent the inhibition due to the slowly equilibrating inhibitory site. Here, we present the first evidence that a compound (3-acetoxy ecgonine methyl ester) exists that prevents inhibition of the slowly equilibrating inhibitory site and that the mechanism of inhibition of this site differs from that of the rapidly equilibrating site. BC3H1 cells containing a fetal mouse muscle-type nAChR were used, and the receptor was activated by carbamoylcholine. The resulting whole-cell current due to the nondesensitized nAChR was determined. Because the nAChR desensitizes rapidly, the measurements required the use of a transient kinetic technique with a time resolution of 10 ms; the cell-flow technique was used. Inhibitors and compounds that alleviate inhibition were tested by determining their effects on the whole-cell current due to activation of the nAChR by carbamoylcholine.     

A novel physiological property of snake bradykinin-potentiating peptides-reversion of MK-801 inhibition of nicotinic acetylcholine receptors

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [相似文献   

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine

Summary 1. Site directed mutagenesis was used to alter the structure ofTorpedo californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed inXenopus laevis oocytes injected within vitro synthesized mRNA and the whole cell currents induced by acetylcholine (ACh) were recorded by two electrode voltage clamp.2. A series of mutations of a highly conserved Arg at position 209 of the subunit ofTorpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (R209K) or His (R209H) at position 209 shifted the EC₅₀ for ACh slightly from 5µM to 12µM and increased the normalized maximal channel activity 8.5-and 3.2-fold, respectively.3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in both wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition.4. Further mutations at different positions of the subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibition whereas mutationsI210A andL212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine.5. These findings support a hypothesis that the quinacrine binding site is located in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the beginning of M1 region might be sheet structure.     

Reversing the action of noncompetitive inhibitors (MK-801 and cocaine) on a protein (nicotinic acetylcholine receptor)-mediated reaction

The nicotinic acetylcholine receptor (nAChR) is one of five structurally related membrane proteins required for communication between approximately 10(12) cells of the mammalian nervous system. The receptor is inhibited by both therapeutic agents and abused drugs. Understanding the mechanism of noncompetitive allosteric inhibitors of the nicotinic acetylcholine receptor is a long-standing and intensely investigated problem. During the past two decades, many attempts have been made to find drugs that prevent cocaine inhibition, including the synthesis of hundreds of cocaine analogues and derivatives, so far without success. The use of newly developed transient kinetic techniques in investigations of the inhibition of the receptor by the anticonvulsant MK-801 [(+)-dizocilpine] and the abused drug cocaine led to an inhibition mechanism not previously proposed. This mechanism indicates the properties of compounds that would prevent allosteric inhibition of the receptor and how to test for such compounds. Here we present the first evidence that small organic compounds (cocaine derivatives) exist that prevent cocaine and MK-801 inhibition of this receptor. These compounds are RTI-4229-70, a previously synthesized cocaine derivative, and based on its structure four newly synthesized cocaine derivatives, RCS-III-143, RCS-III-140A, RCS-III-218, and RCS-III-202A. Because the nAChR desensitizes rapidly, to make the required measurements a cell-flow technique with a time resolution of 10 ms was used to equilibrate BCH(3) cells containing the fetal mouse muscle-type nAChR with carbamoylcholine. The resulting whole-cell current pertaining to the nondesensitized nAChR was determined. Inhibitors and compounds that alleviate inhibition were tested by their effect on the whole-cell current.     

Sensitivity to voltage-independent inhibition determined by pore-lining region of the acetylcholine receptor.

Some noncompetitive inhibitors (e.g., ganglionic blockers) exhibit selectivity for the inhibition of neuronal nicotinic acetylcholine receptors (nAChRs). This study characterizes the mechanism of selective long-term inhibition of neuronal and muscle-neuronal chimeric nAChRs by bis(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (bis-TMP-10 or BTMPS), a bifunctional form of the potent ganglionic blocker tetramethylpiperidine. Long-term inhibition of neuronal nAChRs by bis-TMP-10 has been previously demonstrated to arise, at least in part, from the binding of the bis compound to neuronal beta-subunits. In this study, long-term inhibition is demonstrated to be dependent upon the presence of sequence element(s) within the pore-lining second transmembrane domain (tm2) of neuronal beta-subunits; however, the inhibitor binding site itself does not appear to be contained within the segment of the channel pore influenced by the membrane electric field. Specifically, our results imply that bis-TMP-10 interacts with an activation-sensitive element, the availability of which may be regulated by a sequence in the tm2 domain. Furthermore, we demonstrate a compound length requirement for long-term inhibition that would be consistent with binding to multiple sites located on the extracellular portion of the receptor.     

Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor alpha-subunit by epitope mapping.

Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the alpha-subunit between alpha-156 and alpha-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing alpha-subunit were distinguished in terms of their ability to bind alpha-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10 kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease alpha-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both anti-V8-18 and anti-V8-20 mAbs was determined to be within alpha-156 to alpha-179 by isolation of small immunoreactive peptides from proteolytic digests of the alpha-subunit, while the mAb reactive to V8-10 was bound to an epitope within alpha-339 to alpha-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to alpha-147 to alpha-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease alpha-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of alpha-156 and alpha-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.     

Site of resting state inhibition of the nicotinic acetylcholine receptor by a hydrophobic inhibitor

The lipophilic photoactivatable probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) is a noncompetitive, resting-state inhibitor of the nicotinic acetylcholine receptor (nAChR) that requires tens of milliseconds of preincubation to inhibit agonist-induced cation efflux. At equilibrium, [(125)I]TID photoincorporates into both the ion channel and the lipid-protein interface of the Torpedo nAChR. To determine which of these regions is responsible for resting-state inhibition, we characterized the interactions between [(125)I]TID and nAChR-rich membranes milliseconds after mixing, by use of time-resolved photolabeling. Photolabeling was performed after preincubation times of 2 ms or 600 s (equilibrium), and the efficiencies of incorporation at specific residues were determined by amino-terminal sequence analysis of nAChR-subunit proteolytic fragments isolated by SDS-PAGE and/or reversed-phase HPLC. Equilibration of TID with lipid was complete within a millisecond as determined by both stopped-flow fluorescence quenching of diphenylhexatriene in lipid bilayers and photoincorporation into nAChR-rich membrane phospholipids. Equilibration with the lipid-protein interface (alphaM4) was slightly slower, reaching approximately 50% that at equilibrium after 2 ms preincubation. In contrast, equilibration with the channel region (alpha 2 and deltaM2) was much slower, reaching only 10% that at equilibrium after 2 ms preincubation. Within the ion channel, the ratio of [(125)I]TID incorporation between M2 residues 9', 13', and 16' was independent of preincubation time. We conclude that TID's access to the ion channel is more restricted than to the lipid-protein interface and that TID bound within the ion channel is responsible for flux inhibition upon activation of the nAChR.     

Structure and gating mechanism of the α7 nicotinic acetylcholine receptor

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Mechanism of glutamate receptor-channel function in rat hippocampal neurons investigated using the laser-pulse photolysis (LaPP) technique

Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell. The binding of glutamic acid triggers the channel opening in the microsecond time domain and the reversible inactivation (desensitization) of the receptors in the millisecond time region. The channel-opening mechanism of glutamate receptors was investigated in rat hippocampal neurons voltage-clamped to -60 mV at room temperature and pH 7.4. Two rapid chemical reaction techniques were used: (1) a cell-flow method with a 4-10 ms time resolution to apply L-glutamate and (2) a laser-pulse photolysis technique to release glutamate from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (alphaCNB-caged L-glutamate) with a time constant of 30 micros. The rate and equilibrium constants for channel opening were determined. The results are consistent with the receptor binding two molecules of glutamic acid before the channel opens, with an apparent dissociation constant of 600 microM. Channel opening and closing rate constants, k(op) and k(cl), were determined to be (9.5 +/- 1) x 10(3) s(-1) and (1.1 +/- 0.1) x 10(3) s(-1), respectively. The value of the channel-opening equilibrium constant, Phi (=k(op)/k(cl)), was 8.6 when determined by laser-pulse photolysis and 6.6 in cell-flow experiments. The results suggest that there are at least two forms of glutamate receptors in rat hippocampal neurons that desensitize with different rates. At a concentration of 500 microM glutamate, 80% of the receptors desensitized with a rate of approximately 200 s(-1) and 20% with a rate of approximately 50 s(-1).     

Agrin induces phosphorylation of the nicotinic acetylcholine receptor.

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution.     

On the mechanism of a mammalian neuronal type nicotinic acetylcholine receptor investigated by a rapid chemical kinetic technique. Detection and characterization of a short-lived, previously unobserved, main receptor form in PC12 cells.

The mammalian nicotinic acetylcholine receptor in PC12 cells has many properties characteristic of the neuronal receptors involved in key chemical reactions that are responsible for signal transmission between cells of the nervous system. This report describes initial investigations of the mechanism of this receptor using a rapid chemical kinetic technique with a time resolution of 20 ms, which represents a 250-fold improvement over the best time resolution (5 s) employed in previous studies. Carbamoylcholine, a stable analogue of the neurotransmitter acetylcholine, was the activating ligand used, and the concentration of open transmembrane receptor-channels in PC12 cells was measured by recording whole-cell currents at pH 7.4, 21-23 degrees C, and a transmembrane voltage of -60 mV. Two receptor forms that account for 80% and 20% of the receptor-controlled current were detected; the main receptor form, accounting for 80% of the whole-cell current, desensitized completely before the first measurements had been made in previous studies. Only the main receptor form has been investigated so far using the new method. The constants of a mechanism that accounts for the concentration of the open transmembrane receptor-channel over a 100-fold range of carbamoylcholine concentration were evaluated: the dissociation constant of the site controlling channel opening (K1 = 2.0 mM), the channel-opening equilibrium constant (phi -1 = 5.0), and the dissociation constant of an inhibitory site to which carbamoylcholine binds (KR = 6.5 mM). These evaluated constants allow one to calculate Po, the conditional probability that at a given concentration of carbamoylcholine the receptor-channel is open. Po was also determined in the presence of 2 mM carbamoylcholine by an independent method, the single-channel current-recording technique, and the agreement between the Po values obtained in two independent ways is within experimental error. This result indicates that the time resolution of the chemical kinetic technique employed was sufficient to evaluate the constants pertaining to the active state of the receptor, which forms a transmembrane channel, before its conversion to desensitized receptor forms with different properties. Previous kinetic measurements with a time resolution of 5 s showed that many compounds, such as anesthetic-like molecules, nerve growth factor, and substance P, modify the function of the neuronal receptor in PC12 cells or react specifically with the neuronal but not with the muscle receptor, for example, some toxins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.     

The activation of the nicotinic acetylcholine receptor by the transmitter

Experimental evidence has been published from isolated guinea pig muscle in vitro, and from direct ligand binding to receptors from T. californica, indicating that two agonist ions react with the nicotinic receptor by exchanging for one magnesium ion. It is the basis of the ion exchange receptor pair model, in which two acetylcholine ions exchange for one magnesium ion in contact with and between a pair of negatively charged receptor groups about 4 A apart. In the resting state the electrostatic attraction between the negatively charged receptor groups and the Mg2+ ion exerts a binding force. This binding force is opposed by the quantum mechanical repulsions of the electron clouds of the charged groups and ions in contact, together with the mutual repulsion of the pair of receptor oxyanions. When the Mg2+ ion is replaced by two acetylcholine ions the quaternary heads of the latter are positioned so that they form two mutually repelling ACh+ receptor group dipoles. As the Mg2+ ion leaves, its rehydration energy contributes to the sum of the electron cloud repulsions and the ACh+ receptor group dipole repulsions, causing the receptor groups to be forced apart activating the receptor macromolecule. The subsequent decrease in ACh+ concentration results in the reestablishment of the resting state. The coulombic electrostatic energy, the Born repulsion energy, the London attraction energy and the oxyanion ACh+ dipole repulsion energies have been calculated and shown to be consistent with the model. The displacement of the Mg2+ by two ACh+ ions makes several hundred kcals of energy available for receptor group separation and receptor activation.     

Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit.     

Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.