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.     

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.     

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.     

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.     

Selection of 2′-Fluoro-modified RNA Aptamers for Alleviation of Cocaine and MK-801Inhibition of the Nicotinic Acetylcholine Receptor

The nicotinic acetylcholine receptor (nAChR) belongs to a group of five stracturally related proteins that regulate signal transmission between approximately 10¹² cells of the mammalian nervous system. Many therapeutic agents and abused drugs inhibit the nAChR, including the anti-convulsant MK-801 and the abused drug cocaine. Many attempts have been made to find compounds that prevent inhibition by cocaine. Use of transient kinetic techniques to investigate the inhibition of the receptor by MK-801 and cocaine led to an inhibition mechanism not previously proposed. The mechanism led to the development of combinatorially synthesized RNA ligands that alleviate inhibition of the receptor. However, these ligands are relatively unstable. Here we determined whether much more stable 2-fluoro-modified RNA ligands can be prepared and used to study the alleviation of receptor inhibition. Two classes of 2-fluoro-modified RNA ligands were obtained: One class binds with higher affinity to the cocaine-binding site on the closed-channel form and, as predicted by the mechanism, inhibits the receptor. The second class binds with equal or higher affinity to the cocaine-binding site on the open-channel form and, as predicted by the mechanism, does not inhibit the receptor, and does alleviate cocaine and MK-801 inhibition of the nAChR. The stability of these 2-fluoro-RNAs expands the utility of these ligands.     

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)     

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.     

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)     

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)     

Modulation of MK-801-elicited mouse popping behavior by galantamine is complex and dose-dependent

The ability of phencyclidine (PCP), a noncompetitive antagonist of NMDA receptor-mediated neurotransmission, to precipitate a schizophreniform psychosis in susceptible individuals is consistent with the hypothesized pathologic occurrence of NMDA receptor hypofunction in this disorder. Because the psychosis caused by PCP resembles schizophrenia in all of the relevant domains of psychopathology, investigators have sought to characterize animal models of NMDA receptor hypofunction. MK-801 (dizocilpine) binds to the same hydrophobic channel domain in the NMDA receptor-associated ionophore as PCP, and has been shown to elicit intense irregular episodes of jumping behavior in mice, termed "popping." MK-801-elicited mouse popping is an animal model of NMDA receptor hypofunction that has been used to screen novel candidate compounds for the treatment of schizophrenia. Recently, a selective abnormality in the transduction of the acetylcholine signal at the level of the alpha 7 nicotinic receptor has been described in schizophrenia. The existence of a nicotinic cholinergic abnormality in schizophrenia has stimulated interest in a potential therapeutic role for positive allosteric modulation of nicotinic receptors. Galantamine is a compound that possesses two interesting properties: inhibition of acetylcholinesterase and positive allosteric modulation of nicotinic neurotransmission. Theoretically, galantamine would be expected to increase the efficiency or likelihood that acetylcholine will promote channel opening and ionic conductance at nicotinic receptors. As expected, in the current investigation statistically significant popping behavior was elicited by MK-801 in mice (T(22) = 2.16, P < 0.05). This MK-801-elicited popping was significantly attenuated by 100 mg/kg of galantamine (T(22) = 2.24, P < 0.05). The data show that nicotinic interventions can influence NMDA receptor-mediated neurotransmission in the intact mouse.     

Channel-opening kinetics of GluR6 kainate receptor

GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.     

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   

Time-resolved photolabeling by quinacrine azide of a noncompetitive inhibitor site of the nicotinic acetylcholine receptor in a transient, agonist-induced state

Local anesthetics and other noncompetitive inhibitors (NCIs) of the nicotinic acetylcholine receptor, acting at sites other than the acetylcholine-binding sites, block channel opening and/or cation translation through the open channel. In order to characterize the NCI sites and to decide among possible mechanisms of NCI action, we have photolabeled the receptor in membrane from Torpedo electric tissue with the photolyzable NCI [3H]quinacrine azide ([3H]QA), using a continuous-flow, rapid-mixing device and millisecond-duration irradiation. Membrane, [3H]QA, and effectors were mixed, and, after delay times of 20 ms or greater, the mixture was irradiated for 2 ms, quenched, and collected. Brief exposure of the receptor to acetylcholine, but not to hexamethonium or d-tubocurarine, induced a state particularly susceptible to photoincorporation of [3H]QA. This acetylcholine-induced photoincorporation was exclusively into the alpha and beta chains of the receptor, peaked at 100-ms delay time, declined to 15% of maximum after delay times of minutes, and was blocked by the NCIs proadifen and histrionicotoxin. At 20-ms delay, the dependence of labeling by 2 microM [3H]QA on acetylcholine concentration was characterized by an apparent dissociation constant of about 15 microM and a Hill coefficient of 1. The kinetics of the development of susceptibility to photolabeling and the apparent lack of positive cooperativity in the effect of acetylcholine on this development suggest that the preferentially photolabeled state is a transient, rapidly developing, desensitized state, rather than an open-channel state.     

Minimal RNA Aptamer Sequences That Can Inhibit or Alleviate Noncompetitive Inhibition of the Muscle-Type Nicotinic Acetylcholine Receptor

Combinatorially synthesized nucleotide polymers have been used during the last decade to find ligands that bind to specific sites on biological molecules, including membrane-bound proteins such as the nicotinic acetylcholine receptors (nAChRs). The neurotransmitter receptors belong to a group of four structurally related proteins that regulate signal transmission between ~10¹¹ neurons of the mammalian nervous system. The nAChRs are inhibited by compounds such as the anticonvulsant MK-801 [(+)-dizocilpine] and abused drugs such as cocaine. Based on predictions arising from the mechanism of receptor inhibition by MK-801 and cocaine, we developed two classes of RNA aptamers: class I members, which inhibit the nAChR, and class II members, which alleviate inhibition of the receptor by MK-801 and cocaine. The systematic evolution of ligands by the exponential enrichment (SELEX) method was used to obtain these compounds. Here, we report that we have truncated RNA aptamers in each class to determine the minimal nucleic acid sequence that retains the characteristic function for which the aptamer was originally selected. We demonstrate that a truncated class I aptamer containing a sequence of seven nucleotides inhibits the nAChR and that a truncated class II aptamer containing a sequence of only four nucleotides can alleviate MK-801 inhibition.     

High acetylcholine concentrations cause rapid inactivation before fast desensitization in nicotinic acetylcholine receptors from Torpedo.

By using both a 3 to 4 ms quenched-86Rb+ flux assay and native acetylcholine receptor (AChR) rich electroplaque vesicles on which 50-60% of acetylcholine activation sites were blocked with alpha-BTX, we determined apparent rates of agonist-induced inactivation in AChR from Torpedo under conditions where measured flux response was directly proportional to initial 86Rb+ influx rate. Inactivation kinetics with acetylcholine in both the activating range (10 microM-10 mM) and the self-inhibiting range (15-100 mM) were measured at 4 degrees C. In the presence of 10 microM-1 mM acetylcholine, inactivation is characterized by a single exponential rate constant, kd (fast desensitization). Plots of kd vs. acetylcholine concentration display maximum kds [kd(max)] of 6.6-8.0 s-1, half-maximal kd at 102 +/- 16 microM, and a Hill coefficient of 1.6 +/- 0.3, closely paralleling the initial ion flux response of AChR. Thus, fast desensitization probably occurs from a doubly-liganded preopen state or the open channel state. In the self-inhibiting acetylcholine concentration range, inactivation is biphasic. A "rapid inactivation" phase is complete within 30 ms, followed by fast desensitization at a rate close to kd(max). Both the rate and extent of rapid inactivation increase with acetylcholine concentration, indicating that acetylcholine binds to its self-inhibition site with apparent kon approximately equal to 10(3) M-1s-1 and koff approximately equal to 40 s-1. This slow kon suggests either hindered access to the inhibitory allosteric site or that a fast binding step is followed by a slower conformational change leading to channel inhibition. Overall, our data suggest that acetylcholine binds preferentially to its inhibitory site when the receptor is in the open-channel conformation and that fast desensitization can occur from all multiple-liganded states.     

On the mechanism of the gamma-aminobutyric acid receptor in the mammalian (mouse) cerebral cortex. Chemical kinetic investigations with a 10-ms time resolution adapted to measurements of neuronal receptor function in single cells.

The gamma-aminobutyric acidA (GABA) receptor belongs to a superfamily of proteins involved in chemical reactions that regulate signal transmission between cells of the nervous system and is the target of some of the agents most frequently used in medicine to control disorders of the central nervous system. In contrast to the nicotinic acetylcholine receptor, which initiates signal transmission and is the best characterized member of the superfamily, the GABA receptor forms anion-specific transmembrane channels and inhibits signal transmission. The chemical kinetic experiments described here, in which fast chemical reaction techniques were used, indicate that both receptor proteins may operate by the same mechanism. Also described is the use of a chemical kinetic technique with a 10-ms time resolution that we have developed for making measurements with single cells isolated from specific areas of the nervous system, in this case the cerebral cortex of embryonic mice. A flow device was used to equilibrate receptors on the cell surface with GABA, and the concentration of open transmembrane channels in the cells was then measured by recording the whole-cell currents at pH 7.2, 21-23 degrees C, and a transmembrane voltage of -70 mV. Two different receptor forms, A alpha and A beta, were detected in cerebral cortical cells. Although the ratio of A alpha to A beta varied from cell to cell, on average 35% and 65% of the receptor-controlled current was associated with receptor forms A alpha and A beta, respectively. At saturating concentrations of GABA, the rate coefficients of desensitization, alpha and beta, associated with these two forms have maximal values of 4.4 and 0.7 s-1, respectively. The constants of a mechanism that accounts for the open transmembrane channels of both receptor forms were evaluated over a 50-fold range of GABA concentration. The dissociation constant of the site controlling channel opening was 40 microM for A alpha and 320 microM for A beta. The channel-opening equilibrium constant, phi-1, was 3.5 for A alpha and 20 for A beta. The evaluated constants allow one to calculate Po, the conditional probability that at a given concentration of GABA the receptor-channel is open. Po could also be determined in the presence of 100 microM GABA by an independent method in which different assumptions are made in the interpretation of the experimental results, the single-channel current-recording technique. The value of Po obtained (0.56) was in good agreement with the Po value (0.61) calculated for receptor form A alpha from chemical kinetic measurements at 100 microM GABA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apoptotic Cell Death and Caspase-3 Activation Induced by N-Methyl-d-Aspartate Receptor Antagonists and Their Prevention by Insulin-Like Growth Factor I

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.     

Photolabile protecting groups for an acetylcholine receptor ligand. Synthesis and photochemistry of a new class of o-nitrobenzyl derivatives and their effects on receptor function

Two compounds have been synthesized that feature a photosensitive o-nitrobenzyl moiety attached directly to the carbamate nitrogen of carbamoylcholine. The well-characterized acetylcholine analogue, carbamoylcholine, was released from these derivatives in response to laser light pulses at wavelengths between 300 and 355 nm. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis. The yield of carbamoylcholine molecules per photon absorbed was 0.25. A short-lived photochromic intermediate in the photolysis reaction was detected by laser flash photolysis. A single laser flash induced an instantaneous increase in absorbance at 406 nm, followed by a first-order decay to products, with a half-time of 0.07 ms for one of the compounds [N-[1-(2-nitrophenyl)ethyl]carbamoylcholine iodide] in aqueous buffers at pH 7 and 23 degrees C. Decay rates and quantum yields depended on the nature of the substituent on the protecting group. Evidence is presented in support of the conclusion that the transient species is an aci-nitro intermediate that decays directly to carbamoylcholine and therefore determines its rate of release. The photosensitive carbamoylcholine derivatives activated the nicotinic acetylcholine receptor only after photolysis, as determined by 86Rb+ flux measurements with membrane vesicles prepared from Torpedo californica and Electrophorus electricus. Before photolysis, the compounds interacted weakly with the acetylcholine-binding sites as shown by competitive inhibition of acetylcholine-stimulated flux at high concentrations. The compounds did not induce receptor desensitization at a significant rate. The new compounds afford several major advantages over other photoactivatable acetylcholine analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myasthenic nicotinic receptor mutant interpreted in terms of the allosteric model

An extended Monod—Wymctn—Changeux allosteric-type model is applied to human muscle nicotinic acetylcholine receptors expressed in HEK cells, for both the normal form and the high-affinity human myasthénic mutant, ε T264P. The model is based on a concerted transition between the basal (resting) B state and the active (open-channel) A state, with the equilibrium in the absence of ligand determined by the allosteric constant, L_o = [B₀]/[A₀]. For wild-type receptors the model with L₀ = 910⁸ provides a satisfactory representation of published patch-clamp recordings that yields a distribution of open-channel dwell times with a single peak at 0.7 ms. For the εT264P mutant, the model with L₀ = 100 accounts for the trimodal distribution reported for open-channel dwell times, with peaks at 0.15, 3.8 and 60 ms that correspond to non-, mono- and bi-liganded receptors, respectively. Possible applications of the allosteric model to other myasthénic mutants are considered.     

Inhibition mechanism of rat α₃β₄ nicotinic acetylcholine receptor by the Alzheimer therapeutic tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer's disease, on currents elicited by activation of rat α(3)β(4) nAChR heterologously expressed in KXα3β4R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 ± 0.2 μM and 4.3 ± 1.3 for the channel opening equilibrium constant, Φ(-1). Experiments were performed to investigate whether tacrine is able to activate the α(3)β(4) nAChR. Tacrine did not activate whole-cell currents in KXα3β4R2 cells but inhibited receptor activity at submicromolar concentration. Dose-response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 μM. The increase of Φ(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.