Synthesis, photochemistry, and biological activity of a caged photolabile acetylcholine receptor ligand

A biologically inert photolabile precursor of carbamoylcholine has been synthesized; it is photolyzed to carbamoylcholine, a well-characterized acetylcholine analogue, with a half-time of 40 microseconds at pH 7.0 and a quantum yield of 0.8. The compound, N-(alpha-carboxy-2-nitrobenzyl)carbamoylcholine, was synthesized from (2-nitrophenyl)glycine. The photolysis rates (of five compounds) and the biological activity (of two compounds) were determined, and both properties were found to depend on the nature of the substituents on the photolabile protecting group. Laser pulse photolysis at wavelengths between 308 and 355 nm was used to investigate the wavelength dependence, quantum yield, and rate of the photolysis reaction. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis and by their ability to activate the nicotinic acetylcholine receptor. BC3H1 muscle cells containing those receptors and a cell-flow method were used in the biological assays. The approach described may be useful in the preparation and characterization of other photolabile precursors of neurotransmitters that contain amino groups. The importance of these rapidly photolyzed, inert precursors of neurotransmitters is in chemical kinetic investigations of the reactions involving diverse neuronal receptors; such studies have been hampered because the available techniques have an insufficient time resolution.     

Fourier transform infrared (FTIR) spectroscopic investigation of the nicotinic acetylcholine receptor (nAChR). Investigation of agonist binding and receptor conformational changes by flash-induced release of 'caged' carbamoylcholine.

The binding and interaction of carbamoylcholine with the nicotinic acetylcholine receptor was investigated using photolytically released carbamoylcholine ('caged' carbamoylcholine). Upon UV flash activation of this photolabile substrate analog, characteristic changes in the IR absorbance spectrum were detected. Apart from difference bands arising from the changes of molecular structure upon photolytical release, spectral features can be attributed to the agonist upon binding to the receptor as well as to conformational changes of the receptor itself. The two photo-labile agonist analogs N-[1-(2-nitrophenyl)ethyl] carbamoylcholine iodide (cage I) and N-(alpha-carboxy-2-nitrobenzyl) carbamoylcholine trifluoroacetate (cage II), with different structures for comparison of the 1680-1540 cm-1 region sensitive for protein conformation, yielded consistent results. A preliminary interpretation in terms of substrate binding and local conformational changes of the receptor upon carbamoylcholine binding is provided, in analogy to the binding of acetylcholine, activation, and subsequent deactivation taking place during signal transduction.     

Acetylcholine receptor: evidence for a regulatory binding site in investigations of suberyldicholine-induced transmembrane ion flux in Electrophorus electricus membrane vesicles

Suberyldicholine-induced ion translocation in the millisecond time region in acetylcholine receptor rich membrane vesicles prepared from the electric organ of Electrophorus electricus was investigated in eel Ringer's solution, pH 7.0, 1 degree C. A quench-flow technique with a time resolution of 5 ms was used to measure the transmembrane flux of a radioactive tracer ion (86Rb+). JA, the rate coefficient for ion flux mediated by the active form of the receptor, and alpha, the rate coefficient for the inactivation of the ion flux, increase with increasing suberyldicholine concentrations and reach a plateau value at about 15 microM. At higher suberyldicholine concentrations (greater than 50 microM), a concentration-dependent decrease in the ion flux rate was observed without a corresponding decrease in the rate of receptor inactivation. This regulatory effect was not observed with acetylcholine or carbamoylcholine. The minimal kinetic scheme previously presented for acetylcholine and carbamoylcholine, modified by the inclusion of an additional regulatory ligand-binding site for suberyldicholine and characterized by a single dissociation constant, KR, is consistent with the results obtained over a 10 000-fold concentration range of this ligand. Rate and equilibrium constants pertaining to this scheme were elucidated. Suberyldicholine binds to the regulatory site (KR = 500 microM) approximately 100-fold less well than to its activating sites, and the binding to the regulatory site has no effect on the inactivation (desensitization) rate coefficient alpha [alpha(max) = 5.7 s-1], which is comparable to that observed with acetylcholine. The maximum influx rate coefficient [JA(max) = 18.5 s-1] is approximately twice that obtained when carbamoylcholine is the activating ligand and somewhat higher than when acetylcholine is used.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

How fast does an acetylcholine receptor channel open? Laser-pulse photolysis of an inactive precursor of carbamoylcholine in the microsecond time region with BC3H1 cells.

The integrated function of the nervous system depends on specific and rapid transmission of signals between its constituent cells. The nicotinic acetylcholine receptor is the best known of a group of membrane-bound proteins responsible for such transmission; for this process to occur, a specific neurotransmitter, in this case acetylcholine, must bind to the receptor, which then forms transmembrane channels through which cations pass. The resulting change in transmembrane voltage determines whether or not a signal is transmitted. The question of how fast this process takes place in any neurotransmitter receptor has remained one of the interesting and most challenging in the field. To answer it, many attempts have been made to evaluate the rate constant for the opening of the acetylcholine receptor channel, but in almost all these studies the rate was measured after the receptor-mediated reaction, which involves the open channel and many intermediate states, had reached a quasi equilibrium. This resulted in a plethora of reported values for the rate constant that differ by a factor of up to 50-fold, even when the measurements were made with the same type of cell. The new approach described here involves the use of single cells of a mammalian cell line (BC3H1), containing muscle-type acetylcholine receptors, and the rapid introduction of neurotransmitter to the cell surface. The rapid delivery was achieved by converting a previously synthesized photolabile precursor of carbamoylcholine to carbamoylcholine, a stable amino-group-containing analogue of acetylcholine, with a single laser pulse and an observed photolysis rate of 7300 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photochemistry of allyloxybenzophenones: a pseudo-Paternò-Büchi rearrangement accompanied by hydrogen transfer induced 1,5-cyclization.

The solution photochemistry of the ortho allyloxy-substituted benzophenone has been investigated in detail. Product analysis revealed formation of a diastereomeric mixture of dihydrobenzofuran derivatives by cyclization via a short-lived intermediate 1,5-biradical and an unusual acetal by a pseudo-Paternò-Büchi rearrangement. The latter reaction pathway was supported by means of laser flash photolysis, where a long-lived intermediate with a maximum absorption band at 380 nm was observed. Besides, theoretical calculations (TD-DFT) of this UV-transient resulted in a band with maximum intensity at 390 nm showing a good correlation between experimental results and theoretical calculations. For comparison, the meta-substituent substrate was also investigated showing preferred triplet-triplet energy transfer.     

Rapid laser flash photoaffinity labeling of binding sites for a noncompetitive inhibitor of the acetylcholine receptor

Photoaffinity labeling of the nicotinic acetylcholine receptor from Torpedo marmorata electric tissue was performed in the presence of cholinergic effectors in the millisecond to second time range by a combination of a stopped-flow apparatus and a high-energy pulse laser. The label applied was [3H]triphenylmethylphosphonium, a lipophilic cation previously shown to be a specific blocker of the acetylcholine receptor ion channel. With the receptor in the resting state most of the label was incorporated into the alpha polypeptide chains. In the presence of agonists and antagonists increasing incorporation into the delta- and (less pronounced) the beta-chain was observed. The time course of this increase had a half-life of about 0.4 s, being slower than receptor activation and channel opening. in the resting, active, and even rapidly desensitized state, the alpha polypeptide chains appear to be the primary targets of the photoaffinity reaction. The action spectrum of the photolabeling has a sharp maximum at lambda = 270 nm and a small-side maximum at lambda = 290 nm. It does not resemble the absorption spectrum of the label and may hint at amino acid side chains as the moieties activated by UV light causing the photolabeling. The effector specificity of the observed slow increase of label incorporation into the delta polypeptide chain was investigated. It does not prove that slow desensitization is the underlying event. The agonists acetylcholine and carbamoylcholine as well as treatment of receptor-rich membranes with phospholipase A2 (but not phospholipase D) triggered labeling of delta, but antagonists such as D-tubocurarine and most conspicuously flaxedil had a similar effect.     

Phencyclidine inhibition of the acetylcholine receptor: measurement of cation flux in a sympathetic neuronal cell line using 22Na+ and spectroscopic detection of Cs+

The site of action of phencyclidine, a powerful and increasingly abused drug, in sympathetic nerve cells has not previously been identified. Here it is demonstrated that phencyclidine is a powerful, noncompetitive inhibitor of the nicotinic acetylcholine receptor in a sympathetic nerve cell line, PC-12. In the presence of 1 mM carbamoylcholine the rate of the receptor-controlled influx of 22Na+ is reduced by a factor of 2 by 0.7 microM phencyclidine. Increasing concentrations of carbamoylcholine cannot reverse the inhibitory effect of the drug. Both the transmission of electrical signals between nerve cells and the secretion of catecholamines in the PC-12 cell line depend on the receptor-controlled ion flux. Thus phencyclidine interferes with at least two specific, physiologically important functions of these nerve cells. A new spectroscopic method has been developed to measure cation flux in cells. It is shown that this method can replace measurements of tracer ion flux.     

Nicotinic acetylcholine receptor probed with a photoactivatable agonist: improved labeling specificity by addition of CeIV/glutathione. Extension to laser flash photolabeling.

The molecular structure of Torpedo marmorata acetylcholine binding sites has been investigated previously by photoaffinity labeling. However, besides the nicotine molecule [Middleton et al. (1991) Biochemistry 30, 6987-6997], all other photosensitive probes used for this purpose interacted only with closed receptor states. In the perspective of mapping the functional activated state, we synthesized and developed a new photoactivatable agonist of nAChR capable of alkylation of the acetylcholine (ACh) binding sites, as reported previously [Kotzyba-Hibert et al. (1997) Bioconjugate Chem. 8, 472-480]. Here, we describe the setup of experimental conditions that were made in order to optimize the photolabeling reaction and in particular its specificity. We found that subsequent addition of the oxidant ceric ion (CeIV) and reduced glutathione before the photolabeling step lowered considerably nonspecific labeling (over 90% protection with d-tubocurarine) without affecting the binding properties of the ACh binding sites. As a consequence, irradiation at 360 nm for 20 min in these new conditions gave satisfactory coupling yields (7.5%). A general mechanism was proposed to explain the successive reactions occurring and their drastic effect on the specificity of the labeling reaction. Last, these incubation conditions can be extended to nanosecond pulsed laser photolysis leading to the same specific photoincorporation as for usual irradiations (8.5% coupling yield of ACh binding sites, 77% protection with carbamylcholine). Laser flash photocoupling of a diazocyclohexadienoyl probe on nAChR was achieved for the first time. Taken together, these data indicate that future investigation of the molecular dynamics of allosteric transitions occurring at the activated ACh binding sites should be possible.     

Riboflavin-sensitized photooxidation of isohumulones and derivatives.

Isohumulones, the bitter principles in beer, are decomposed by light-induced reactions, thereby adversely affecting beer quality. Upon absorption of visible light, riboflavin is excited and interacts with isohumulones, as well as with oxidized and reduced derivatives thereof. Reaction kinetics were investigated by laser flash photolysis at 355 nm and at 440 nm, and analysis of kinetic data afforded detailed insights into the reaction mechanism.     

Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site

The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.     

Ligand-induced conformation changes in Torpedo californica membrane-bound acetylcholine receptor

A time-dependent increase in ligand affinity has been studied in cholinergic ligand binding to Torpedocalifornica acetylcholine receptor by inhibition of the kinetics of of [125I]-alpha-bungarotoxin-receptor complex formation. The conversion of the acetylcholine receptor from low to high affinity form was induced by both agonists and antagonists of acetylcholine and was reversible upon removal of the ligand. The slow ligand induced affinity change in vitro resembled electrophysiological desensitization observed at the neuromuscular junction and described by a two-state model (Katz, B., & Thesleff, S. (1957) J. Physiol. 138, 63). A quantitative treatment of the rate and equilibrium constants determined for binding of the agonist carbamoylcholine to membrane bound acetylcholine receptor indicated that the two-state model is not compatible with the in vitro results.     

Acetylcholine receptor: channel-opening kinetics evaluated by rapid chemical kinetic and single-channel current measurements.

A combination of rapid chemical kinetic (quench-flow) and single-channel current measurements was used to evaluate kinetic parameters governing the opening of acetylcholine-receptor channels in the electric organ (electroplax) of Electrophorus electricus. Chemical kinetic measurements made on membrane vesicles, prepared from the E. electricus electroplax, using carbamoylcholine (200 microM-20 mM) at 12 degrees C, pH 7.0, and in the absence of a transmembrane voltage, yielded values for K1 (dissociation constant for receptor activation), phi (channel closing equilibrium constant), J (specific reaction rate for ion flux), and alpha max (maximum inactivation rate constant) of 1 mM, 3.4, 4 x 10(7) M-1 s-1, and 12 s-1, respectively. The single-channel current recordings were made with cells also from the E. electricus electroplax, at the same temperature and pH as the chemical kinetic measurements, using carbamoylcholine (50 microM-2 mM), acetylcholine (500 nM), or suberyldicholine (20 nM). Single-channel current measurements indicated the presence of a single, unique open-channel state of the E. electricus receptor, in concurrence with previous, less extensive measurements. The rate constant for channel closing (kc) obtained from the mean open time of the receptor channel is 1,100 s-1 for carbamoylcholine, 1,200 s-1 for acetylcholine, and 360 s-1 for suberyldicholine at zero membrane potential; and it decreases e-fold for an 80 mV decrease in transmembrane voltage in each case. The decrease in mean open times of the receptor channel that is associated with increasing the carbamoylcholine concentration is interpreted to be due to carbamoylcholine binding to the regulatory (inhibitory) site on the receptor. An analysis of data obtained with carbamoylcholine showed that the closed times within a burst of channel activity fit a two-exponential distribution, with a concentration-independent time constant considered to be the time constant for carbamoylcholine to dissociate from the regulatory site, and a carbamoylcholine concentration-dependent, but voltage-independent, time constant interpreted to represent the rate constant for channel opening (k0). An analysis of the mean closed time data on the basis of the minimum model gives values for K1 and k0 of 0.6 mM and 440 s-1, respectively, with carbamoylcholine as the activating ligand. The values obtained for K1, phi (= kc/k0), J, and alpha from the single-channel current measurements using electroplax are in good agreement with the values obtained from the chemical kinetic measurements using receptor-rich vesicles prepared from the same cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.     

Photochemical reactions of 4-thiouridine disulfide and 4-benzylthiouridine--the involvement of the 4-pyrimidinylthiyl radical.

The reactions of a disulfide and a benzylsulfide derived from 4-thiouridine were studied in aqueous acetonitrile using stationary and laser flash photolysis methods. Irradiation of the compounds results in specific cleavage of the S-S bond in the disulfide and the S-CH(2) bond in the sulfide. Identical pyrimidine-derived intermediates were observed in the transient absorption spectra (lambda(max) = 420 nm, epsilon(max) approximately 2500 M(-1) cm(-1)) recorded for both compounds in laser flash photolysis experiments. The intermediate was identified as the 4-pyrimidinylthiyl radical. Irradiation of the disulfide in the absence of oxygen gives 4-thiouridine while the sulfide under identical conditions produced, additionally, 3-benzyl-4-thiouridine as a stable photoproduct. The formation of the latter photoproduct provides evidence for the existence of the N-centered 4-thioxopyrimidynyl radical formed from the initially produced S-centered (thiyl) radical. The 4-thiouridine is formed from the radicals generated in the primary photochemical step by an H abstraction reaction from the solvent (acetonitrile) or from additives (alcohols) that were purposely added. Interestingly, in contrast to the benzylsulfide, the photoreaction of the disulfide is quenched by molecular oxygen with the concomitant formation of uridine. However it appears that uridine is not produced as a result of the reaction of the radicals with oxygen. A mechanism is proposed for the photochemical transformations of the disulfide and benzylsulfide derived from 4-thiouridine. The proposed mechanism is based on the structures of the identified stable photoproducts, the values of the photoreaction quantum yields determined under differing irradiation conditions, and the flash photolysis results.     

Ligand binding to cytochrome c peroxidase from Pseudomonas aeruginosa

The high potential heme site of Pseudomonas cytochrome c peroxidase has His and Met as ligands. On reduction, the Fe-met bond becomes photosensitive. Following photolysis, the bond reforms with a half-time of 35 ps. The low potential heme peroxidatic site of the fully reduced enzyme has been shown to bind to a range of ligands. The compounds with carbon monoxide, methyl, ethyl, n-butyl, and t-butyl isonitriles have been investigated by laser flash photolysis. All are photosensitive and show different degrees of geminate recombination of ligand in the picosecond and nanosecond time ranges. Carbon monoxide shows the least effect. The three straight-chain isonitriles show about 50% geminate recombination with half-times of the order of 10 ns. t-Butyl isonitrile shows more and faster recombination. These results imply considerable freedom of movement within the active site for the smaller ligands.     

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)     

Studies of the primary oxygen intermediate in the reaction of fully reduced cytochrome oxidase.

The formation and disappearance of a photosensitive species during the reaction of reduced cytochrome c oxidase (putatively a3II.O2), EC 1.9.3.1, has been followed by (a) mixing a3II.CO with O2 in a stopped flow apparatus; (b) initiating the oxygen-oxidase reaction by removing CO with a laser flash; (c) probing the reaction mixture for photosensitivity with a second laser flash. Photosensitivity appears in the reaction mixture after the first laser flash, reaches a maximum after 50-60 microseconds ([O2] greater than 100 microM), and disappears in a further 50-100 microseconds. The kinetics can be represented by the scheme [formula: see text]. In species B, O2 is associated with the protein, possibly CuB, but not with the heme. Species C is the photosensitive a3II.O2 complex, and in D, a3 iron has been oxidized. The formation of species C is responsible for the rapid phase of absorbance change in the oxidase-oxygen reaction. The rate of reaction with oxygen approaches the limit of 35,000 s-1 at high oxygen. Nitric oxide, however, reacts with FeII oxidase with a rate of 1 x 10(8) M-1 s-1, which is accurately maintained up to an observed rate of 10(5) s-1. In flash photolysis experiments, approximately half of the photodissociated nitric oxidase recombines in a biphasic geminate reaction with rates of 1 x 10(8) s-1 and 1 x 10(7) s-1.     

Mechanism-based approach to the successful prevention of cocaine inhibition of the neuronal (alpha 3 beta 4) nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) belongs to a family of five channel-forming proteins that regulate communication between the approximately 10(12) cells of the nervous system. A minimum mechanism of inhibition of the muscle-type nAChR (1) by the noncompetitive inhibitors cocaine and MK-801 [(+)-dizocilpine, an anticonvulsant] indicated they bind to a regulatory site, with higher affinity for the closed-channel form than for the open-channel form, thus shifting the equilibrium toward the closed-channel form and inhibiting receptor function. The mechanism predicts that compounds that bind to this regulatory site with equal or higher affinity for the open-channel conformation than for the closed-channel conformation will prevent receptor inhibition (1). Does a neuronal form of the receptor behave similarly? The mechanism of inhibition of the neuronal nAChR by cocaine and MK-801 using rapid chemical kinetic techniques was investigated. The alpha3beta4 nAChR stably expressed in HEK 293 cells was used in these investigations. Whole-cell currents originated from a major and minor nAChR isoform. Only the major isoform has been characterized. For the dominant, rapidly desensitizing isoform, the carbamoylcholine dissociation constant for the site controlling receptor activation, Kd, is 2 mM; the channel-opening equilibrium constant, Phi(-1), is 4; and the dominant desensitization rate constant, k34, is 20 s(-1). Cocaine inhibits the receptor noncompetitively, with an apparent KI of 84 and 26 microM at high and low carbamoylcholine concentrations, at which concentrations the receptor is mainly in the open- or closed-channel form, respectively. Similar results were obtained with MK-801. A combinatorially synthesized RNA ligand and a cocaine analogue alleviated cocaine inhibition of this neuronal receptor.     

On the mechanism of inhibition of the nicotinic acetylcholine receptor by the anticonvulsant MK-801 investigated by laser-pulse photolysis in the microsecond-to-millisecond time region.

The mechanism of inhibition of the muscle nicotinic acetylcholine receptor is of interest because of the many drugs which are known to modify its function. The laser-pulse photolysis technique, using a photolabile, biologically inert ligand (caged carbamoylcholine) for the nicotinic acetylcholine receptor, and BC3H1 cells have been used to investigate the mechanism of inhibition of the receptor by MK-801 [(+)-dizocilpine] in the microsecond-to-millisecond time region. MK-801 is an anticonvulsant and a known inhibitor of the N-methyl-D-aspartate and nicotinic acetylcholine receptors. Both the chemical kinetic and the single-channel current-recording measurements reported here indicate the existence of two inhibition processes, one occurring within 50 ms and the other within about 1 s of equilibration of the receptor with the inhibitor. Unless stated otherwise, here we characterize the receptor inhibition observed when MK-801 is equilibrated with the receptor for only 50 ms. We determined the effect of MK-801 on the concentration of the open receptor-channels and the apparent dissociation constant of the inhibitor from the closed-channel (KI(obs) = 180 microM) and open-channel ( = 950 microM) forms. Within a few milliseconds after inhibitor binding, decreases to about 100 microM, due to an inhibitor-induced isomerization to an inactive receptor form. A mechanism that incorporates the new results is proposed. It includes the formation of an ion-conducting receptor:inhibitor complex with a channel-opening equilibrium constant that is unfavorable compared to the open-channel receptor form in the absence of inhibitor. In the MK-801 concentration range of 0-500 microM, this mechanism accounts for the observed MK-801-induced decrease in the concentration of open channels. At high concentrations of carbamoylcholine, when the receptor is mainly in the open-channel form, the conducting receptor:inhibitor complex isomerizes to a nonconducting state with a rate constant of about 2400 s-1 for the forward reaction and 230 s-1 for the back reaction. It is shown that the proposed new mechanism, based on transient kinetic measurements, also accounts for the results of previous investigations with other inhibitors (procaine, cocaine), which were carried out under both pre-steady-state and equilibrium conditions. A compound that binds to the same regulatory site on the receptor as MK-801 but does not affect the channel-opening equilibrium constant may have considerable use in protecting an organism from the effects of abused drugs.