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)     

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)     

Chemical kinetic measurements of the effect of trans- and cis-3,3'-Bis[(trimethylammonio)methyl]azobenzene bromide on acetylcholine receptor mediated ion translocation in Electrophorus electricus and Torpedo californica

A quench-flow technique was used to study the effect of trans- and cis-3,3'-bis[(trimethylammonio)methyl]azobenzene bromide (trans- and cis-Bis-Q), photoisomerizable ligands, on the acetylcholine receptor in vesicles prepared from the electric organ of Electrophorus electricus and of Torpedo californica. In E. electricus, two rate coefficients of the receptor-mediated translocation of 86Rb+ induced with trans-Bis-Q were measured: JA, the rate coefficient for ion flux, and alpha, the rate coefficient for receptor inactivation (desensitization). Both rate coefficients increase with increasing concentrations of Bis-Q up to 50 microM. At higher concentrations JA decreases in a concentration-dependent manner while alpha remains unchanged. This effect was previously observed with suberyldicholine [Pasquale, E. B., Takeyasu, K., Udgaonkar, J., Cash, D.J., Severski, M.C., & Hess, G. P. (1983) Biochemistry 22, 5967-5973] and with acetylcholine [Takeyasu, K., Udgaonkar, J., & Hess, G. P. (1983) Biochemistry 22, 5973-5978] and was analyzed in terms of a minimum mechanism that accounts for the properties of activation, desensitization, and inhibition of the receptor. Two molecules of trans-Bis-Q must be bound for the channel to open, but at concentrations greater than 50 microM the population of open channels decreases because of the additional binding of one molecule of trans-Bis-Q to a regulatory inhibitory site, independent of the activating sites. cis-Bis-Q does not induce transmembrane ion flux, but it does inhibit the response of the receptor to acetylcholine and induces inactivation (desensitization) in the micromolar range.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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.     

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)     

Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

Noncompetitive inhibition of acetylcholine receptor-controlled ion translocation was studied in membrane vesicles prepared from both Torpedo californica and Electrophorus electricus electroplax. Ion flux was measured in the millisecond time region by using a spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, and a quench-flow technique using 86Rb+. The rate coefficient of ion flux prior to receptor inactivation (desensitization), JA, was measured at different acetylcholine and inhibitor concentrations, in order to assess which active (nondesensitized) receptor forms bind noncompetitive inhibitors. The degree of inhibition of JA by the inhibitors studied (cocaine, procaine, and phencyclidine) was found to be independent of acetylcholine concentration. The results are consistent with a mechanism in which each compound inhibits by binding to a single site that exists with equal affinity on all active receptor forms. Mechanisms in which the inhibitors bind exclusively to the open-channel form of the receptor are excluded by the data. The same conclusions were reached in cocaine experiments at 0-mV and procaine experiments at -25-mV transmembrane voltage in T. californica vesicles. It had been previously shown that phencyclidine, in addition to decreasing JA (by binding to active receptors), also increases the rate of rapid receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptors (by binding better to inactivated receptor than to active receptor in the closed or open conformations). These effects were not observed with cocaine or procaine. Here it is shown that despite these differential effects on inactivation, cocaine and phencyclidine bind to the same inhibitory site on active receptors (in E. electricus vesicles).(ABSTRACT TRUNCATED AT 250 WORDS)     

Curare binding and the curare-induced subconductance state of the acetylcholine receptor channel.

The curare-induced subconductance state of the nicotinic acetylcholine receptor (AChR) of mouse skeletal muscle was examined using the patch-clamp technique. Two mechanisms for the generation of subconductance states were considered. One of these mechanisms entails allosteric induction of a distinct channel conformation through the binding of curare to the agonist binding site. The other mechanism entails the binding of curare to a different site on the protein. Occupation of this site would then limit the flow of ions through the channel. The voltage dependence and concentration dependence of subconductance state kinetics are consistent with curare binding to a site within the channel. The first order rate constant for binding is 1.2 X 10(6) M-1s-1 at 0 mV, and increases e-fold per 118 mV of membrane hyperpolarization. The rate of curare dissociation from this site is 1.9 X 10(2)s-1 at 0 mV, and decreases e-fold per 95 mV hyperpolarization. The equilibrium constant is 1.4 X 10(-4) M at 0 mV, and decreases e-fold per 55 mV hyperpolarization. This voltage dependence suggests that the fraction of the transmembrane potential traversed by curare in binding to this site is 0.46 or 0.23, depending on whether one assumes that one or both charges of curare sense the electric field. Successive reduction and alkylation of the AChR agonist binding sites with dithiothreitol (DTT) and N-ethyl maleimide (NEM), a treatment which results in the loss of responsiveness of the AChR to agonists, produced no change in curare-induced subconductance events, despite the fact that after this treatment most of the channel openings occurred spontaneously. Mixtures of high concentrations of carbamylcholine (CCh) with a low concentration of curare, which produce channel openings gated predominantly by CCH, resulted in subconductance state kinetics similar to those seen in curare alone at the same concentration. Thus displacement by CCh of curare from the agonist binding sites does not prevent curare from inducing subconductances. The results presented here support the hypothesis that curare induces subconductance states by binding to a site on the receptor other than the agonist binding sites, possibly within the channel pore. It is the occupation of this site by curare that limits the flow of ions through an otherwise fully opened channel.     

Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.     

Acetylcholine receptor inhibition by d-tubocurarine involves both a competitive and a noncompetitive binding site as determined by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

The issue of whether d-tubocurarine, the classical acetylcholine receptor inhibitor, inhibits the receptor by a competitive or noncompetitive mechanism has long been controversial. d-Tubocurarine, in this study, has been found to be both a competitive (KC = 120 nM) and a noncompetitive (KNC = 4 microM) inhibitor of receptor-mediated ion flux at zero transmembrane voltage in membrane vesicles prepared from Electrophorus electricus electroplax. A spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, was used to measure both the rate coefficient of ion flux prior to receptor inactivation (desensitization) and the rate coefficient of the rapid inactivation process. Inhibition by d-tubocurarine of the initial rate of ion flux decreased with increasing acetylcholine concentration, consistent with competitive inhibition, but the inhibition by micromolar concentrations of d-tubocurarine could not be overcome with saturating concentrations of acetylcholine, consistent with noncompetitive inhibition. A minimum mechanism is proposed in which d-tubocurarine competes for one of the two acetylcholine activating sites and also binds to a noncompetitive site. The present data do not distinguish between one or two competitive sites, although one successfully accounts for all of the data. By variation of the acetylcholine concentration, the two types of sites could be studied in isolation. Binding of d-tubocurarine to the noncompetitive site does not change the rate of rapid receptor inactivation, whereas binding of d-tubocurarine to the competitive site decreases the rate of rapid inactivation by displacing acetylcholine, in agreement with the observation that d-tubocurarine does not inactivate (desensitize) the E. electricus receptor by itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cadmium block of squid calcium currents. Macroscopic data and a kinetic model

The mechanism of Cd2+ block of Ca2+ currents (ICa) was explored in squid neurons using whole-cell patch clamp. Control currents activated sigmoidally, more rapidly at more positive potentials, and did not inactivate significantly. External Cd2+ up to 250 microM reduced ICa reversibly. For small depolarizations, the current for a step of 10 ms increased to a maintained value, resembling the control; but for Vm greater than 0 mV, the increase was followed by a decrease, as Cd2+ block became greater. Final block was greater for larger depolarizations. At 0 mV the half-blocking concentration was 125 microM. Tail currents, measured as channels close, had an initial "hook" when recorded in Cd2+: currents increased transiently, then decreased. This suggests that Cd2+ escapes from some channels, which then conduct briefly before closing. Analysis of tail currents shows that Cd2+ does not slow channel closing. The data can be explained if Cd2+ is a permeant blocker of Ca2+ channels and if channels can close when occupied by Cd2+. Cd2+ permeates the channels, but binds transiently to a site in the pore, obstructing the passage of other ions (e.g., Ca2+). Dwell time depends on the transmembrane potential, becoming shorter for more negative internal potentials. A five-state model was used to simulate the steady-state and kinetic features. It combines a Hodgkin-Huxley type m2 gating scheme and a one-site Woodhull ionic blockage model for a permeant blocker and includes a closed blocked state. To fit the data, the binding site for Cd2+ had to be near the outer end of the pore, with a well depth of -12.2 RT, and with a barrier at each end of the pore. The model predicts that the Cd2+ entry rate is nearly voltage independent, but the exit rate is steeply voltage dependent (e-fold/17 mV). Analysis further suggests that the channel closes at a normal rate with Cd2+ in the pore.     

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.     

Calcium channel block by (-)devapamil is affected by the sequence environment and composition of the phenylalkylamine receptor site.

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.     

Measurement of plasma membrane potential in isolated rat hepatocytes using the lipophilic cation, tetraphenylphosphonium: correction of probe intracellular binding and mitochondrial accumulation.

The lipophilic cation tetraphenylphosphonium (TPP+) has been extensively utilized as the probe for the membrane potential (Vm) in various cells. For application to mammalian cells, however, two serious problems require resolution: (1), correction of TPP+ binding to intracellular constituents and (2), estimation of the considerable TPP+ accumulation in mitochondria. We propose here a simple corrective method for the TPP+ binding and its accumulation. TPP+ distribution is assumed as: (1), two compartments (a cytosolic and a mitochondrial space); (2), a proportional relationship between TPP+ bound amount and its unbound concentration in each compartment. We theoretically derived the simple equation: Vm = - RT/F ln(C/Mphys ratio/C/Mabol ratio) where R, T and F have their usual thermodynamic significance. Here, the C/M ratio is defined as the ratio of TPP+ concentration of apparent intracellular to extracellular space. The suffixes phys and abol, respectively, mean the physiological and solely Vm-abolished conditions. This equation was checked with hepatocytes, because estimating hepatocytes Vm with TPP+ distribution is not considered possible because of the relatively high mitochondrial content. The selective Vm abolition was achieved by permeabilization with 20 microM of amphotericin B. The Vm value was, thus, estimated to be -38.6 +/- 0.3 mV, compatible with those obtained with microelectrodes in other laboratories. Vm in hepatocytes is composed of transmembrane K+ diffusion potential (-20.6 +/- 0.3 mV) and electrogenic Na+/K(+)-ATPase (-19.6 +/- 0.4 mV). Addition of rheogenic L-alanine caused a transient but significant depolarization (from control to -34 +/- 0.3 mV). These results taken together indicate that hepatocyte Vm can be accurately determined with the present simple method, so that it may possibly be applicable to the evaluation of Vm in other mammalian cells.     

Interaction of Ba2+ with the pores of the cloned inward rectifier K+ channels Kir2.1 expressed in Xenopus oocytes.

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.     

Inhibition of the serotonin 5-HT3 receptor by nicotine, cocaine, and fluoxetine investigated by rapid chemical kinetic techniques.

The 5-HT(3) serotonin receptor plays an important role in regulating communication between cells in the central and peripheral nervous systems. It is the target of many different therapeutic agents and abused drugs. A rapid chemical kinetic method with a time resolution of 10 ms in combination with the whole-cell current-recording technique was employed to study the receptor in NIE-115 mouse neuroblastoma cells. The mechanism of the channel-opening process, receptor desensitization, and receptor inhibition by nicotine, cocaine, and fluoxetine were investigated. Two different forms of the 5-HT(3) serotonin receptor, each with a different desensitization rate, were observed. The inhibition of the receptor by nicotine has not previously been reported. Both nicotine and cocaine compete with serotonin for the receptor site that controls channel opening, with observed dissociation constants of 25 and 7 microM, respectively. Fluoxetine (Prozac), a widely used antidepressant, occupies a different regulatory site on the receptor with an apparent K(i) value of 244 microM.     

Site-selective agonist binding to the nicotinic acetylcholine receptor from Torpedo californica

Fluorescent energy transfer measurements of dansyl-C6-choline binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica were used to determine binding characteristics of the alpha gamma and alpha delta binding sites. Equilibrium binding measurements show that the alpha gamma site has a lower fluorescence than the alpha delta site; the emission difference is due to differences in the intrinsic fluorescence of the bound fluorophores rather than differences in energy transfer at the two sites. Stopped-flow fluorescence kinetics showed that dissociation of dansyl-C6-choline from the AChR in the desensitized conformation occurs 5-10-fold faster from the alpha gamma site than from the alpha delta site. The dissociation rates are robust for distinct protein preparations, in the presence of noncompetitive antagonists, and over a broad range of ionic strengths. Equilibrium fluorescent binding measurements show that dansyl-C6-choline binds with higher affinity to the alpha delta site (K = 3 nM) than to the alpha gamma site (K = 9 nM) when the AChR is desensitized. Similar affinity differences were observed for acetylcholine itself. The distinct dissociation rates permit the extent of desensitization to be measured at each site during the time course of binding. This sequential mixing method of measuring the desensitized state population at each agonist site can be applied to study the mechanism of AChR activation and subsequent desensitization in detail.     

Block by acetylcholine of mouse muscle nicotinic receptors,stably expressed in fibroblasts

We have measured the concentration and voltage dependence of block by acetylcholine (ACh) of fetal- and adult-type mouse muscle nicotinic receptors, expressed in a fibroblast cell line. Data, obtained at a transmembrane potential of -60 mV and with ACh concentrations of 1 mM and above, are broadly consistent with the occlusion of an open channel with a single ACh+ ion (simple open channel block). The rate of recovery from block is approximately 40,000s-1 and has only a weak voltage dependence. This is in contrast to the strong voltage dependence observed for the degree of block. Deviations from the predictions of the simple model are seen in data collected at positive transmembrane potentials and at negative potentials for ACh concentrations < 1 mM. Less concentration dependence is observed than expected. Of a number of models tested, we demonstrate that two models incorporating both a high and a low affinity blocking site can predict our data.     

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.