Hydroxylated residues influence desensitization behaviour of recombinant alpha3 glycine receptor channels

The human glycine receptor subunit alpha3 exists in two splice variants (alpha3K/L), with alpha3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3-4 loop. Homomeric alpha3K glycine receptors show faster desensitization than alpha3L receptors. Ion channel properties were compared of alpha3L, alpha3K, and of the triple mutant alpha3LDeltaOH = alpha3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site-directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch-clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non-desensitizing current was 68 +/- 13% for alpha3L, 21 +/- 13% for alpha3K, and 48 +/- 16% for alpha3LDeltaOH. Desensitization time constants at saturating glycine concentration were 8.4 +/- 2.8 s, 1.9 +/- 2.3 s, and 2.8 +/- 0.4 s, for alpha3L, alpha3K, and the triple mutant alpha3LDeltaOH, respectively. In contrast, single-channel and whole-cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3-4 loop of the glycine receptor alpha3 subunit are crucial, but not exclusive, determinants of receptor desensitization.     

Glycine-dependent activation of NMDA receptors

N-methyl-d-aspartate (NMDA) receptors are the only neurotransmitter receptors whose activation requires two distinct agonists. Heterotetramers of two GluN1 and two GluN2 subunits, NMDA receptors are broadly distributed in the central nervous system, where they mediate excitatory currents in response to synaptic glutamate release. Pore opening depends on the concurrent presence of glycine, which modulates the amplitude and time course of the glutamate-elicited response. Gating schemes for fully glutamate- and glycine-bound NMDA receptors have been described in sufficient detail to bridge the gap between microscopic and macroscopic receptor behaviors; for several receptor isoforms, these schemes include glutamate-binding steps. We examined currents recorded from cell-attached patches containing one GluN1/GluN2A receptor in the presence of several glycine-site agonists and used kinetic modeling of these data to develop reaction schemes that include explicit glycine-binding steps. Based on the ability to match a series of experimentally observed macroscopic behaviors, we propose a model for activation of the glutamate-bound NMDA receptor by glycine that predicts apparent negative agonist cooperativity and glycine-dependent desensitization in the absence of changes in microscopic binding or desensitization rate constants. These results complete the basic steps of an NMDA receptor reaction scheme for the GluN1/GluN2A isoform and prompt a reevaluation of how glycine controls NMDA receptor activation. We anticipate that our model will provide a useful quantitative instrument to further probe mechanisms and structure–function relationships of NMDA receptors and to better understand the physiological and pathological implications of endogenous fluctuations in extracellular glycine concentrations.     

Mechanism of agonist and antagonist binding to alpha 2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex

The alpha 2 adrenergic receptor (AR) inhibits adenylate cyclase via an interaction with Ni, a guanine nucleotide binding protein. The early steps involved in the activation of the alpha 2 AR by agonists and the subsequent interaction with Ni are poorly understood. In order to better characterize these processes, we have studied the kinetics of ligand binding to the alpha 2 AR in human platelet membranes on the second time scale. Binding of the alpha 2 antagonist [3H]yohimbine was formally consistent with a simple bimolecular reaction mechanism with an association rate constant of 2.5 X 10(5) M-1 s-1 and a dissociation rate constant of 1.11 X 10(-3) s-1. The low association rate constant suggests that this is not a diffusion-limited reaction. Equilibrium binding of the alpha 2 adrenergic full agonist [3H]UK 14,304 was characterized by two binding affinities: Kd1 = 0.3-0.6 nM and Kd2 = 10 nM. The high-affinity binding corresponds to approximately 65% and the low-affinity binding to 35% of the total binding. The kinetics of binding of [3H]UK 14,304 were complex and not consistent with a mass action interaction at one or more independent binding sites. The dependence of the kinetics on [3H]UK 14,304 concentration revealed a fast phase with an apparent bimolecular reaction constant kappa + of 5 X 10(6) M-1 s-1. The rate constants and amplitudes of the slow phase of agonist binding were relatively independent of ligand concentration. These results were analyzed quantitatively according to several variants of the "ternary complex" binding mechanism. In the model which best accounted for the data, (1) approximately one-third of the alpha 2 adrenergic receptor binds agonist with low affinity and is unable to couple with a guanine nucleotide binding protein (N protein), (2) approximately one-third is coupled to the N protein prior to agonist binding, and (3) the remainder interacts by a diffusional coupling of the alpha 2 AR with the N protein or a slow, ligand-independent conformational change of the alpha 2 AR-N protein complex. The rates of interaction of liganded and unliganded receptor with N protein are estimated.     

How fast does the gamma-aminobutyric acid receptor channel open? Kinetic investigations in the microsecond time region using a laser-pulse photolysis technique.

The gamma-aminobuytric acid(A) (GABA(A)) receptor is a membrane-bound protein that mediates signal transmission between neurons through formation of chloride ion channels. GABA is the activating ligand, which upon binding to the receptor triggers channel opening in the microsecond time domain and reversible desensitization of the receptor in the millisecond time region. We have investigated the channel-opening mechanism for this receptor in rat hippocampal neurons before the protein desensitizes by using a rapid flow method (cell-flow) with a 10 ms time resolution and a laser-pulse photolysis technique with a approximately 30 micros time resolution to determine the rate and equilibrium constants for channel opening and closing. Two different forms of the receptor, namely, a rapidly and a slowly desensitizing form, exist in the rat hippocampal cells and are characterized by their different rates for desensitization. At 250 microM GABA the rate constant for desensitization was 2.3 +/- 0.4 s(-)(1) for the rapidly desensitizing form and 0.4 +/- 0.1 s(-)(1) for the slowly desensitizing form. The dissociation constant of GABA from the site controlling channel opening was 100 +/- 40 microM for the rapidly desensitizing form and 120 +/- 60 microM for the slowly desensitizing form. The rate constants for channel closing did not differ significantly for the two forms, 85 +/- 20 s(-)(1) for the rapidly desensitizing and 100 +/- 60 s(-)(1) for the slowly desensitizing form. However, the channel-opening rate constant differed by a factor of 3, 1840 +/- 160 s(-)(1) for the rapidly desensitizing and 6700 +/- 330 s(-)(1) for the slowly desensitizing form. This difference in the rate constant for channel opening for the two forms, determined by the laser-pulse photolysis technique, is reflected as a shift in the channel-opening equilibrium constant, which is 7 +/- 5 and 20 +/- 15 for the rapidly and slowly desensitizing forms respectively, determined by the cell-flow method. These constants, together with the concentration of GABA and the concentration of receptor sites in the membrane, determine the number of channels that open as a function of GABA concentration, and the rate at which they open and close. These constants play an important role in determining the rate of the transmembrane ion flux and, therefore, the receptor-controlled changes in transmembrane voltage that trigger signal transmission.     

Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor

Fluorescence correlation spectroscopy (FCS) is suited to determine low concentrations (10(-8) M) of slowly interacting molecules with different translational diffusion coefficients on the level of single molecule counting. This new technique was applied to characterize the interaction dynamics of tetramethylrhodamin labelled alpha-bungarotoxin (B( *)) with the detergent solubilized nicotinic acetylcholine receptor (AChR) of Torpedo californica electric organ. At pseudo-first-order conditions for AChR, the complex formation with B( *) is monophasic. The association rate coefficient of the monoliganded species AChR . B is k(ass)' = 3.8 . 10(3) s(-1) at 293 K (20 degrees C). The dissociation of bound B( *) from the monomer species AChR . B( *) . B (and AChR . B(2)( *)), initiated by adding an excess of nonlabelled alpha-bungarotoxin (B), is biphasic suggesting a three state cascade for the B-sites: R(alpha) --> R(alpha)' --> R(alpha)' with the exchange dissociation constants: (k(diss)')(B) = 5.5(+/-1) . 10(-5) s(-1) and (k(diss)')(B) = 3(+/-1) . 10(-6) s(-1) at 293 K. The data are consistent with dissociative intermediate steps of ligand exchange on two different interconvertible conformations of one binding site. The dissociation of bound B( *) by excess of the neurotransmitter acetylcholine (ACh) is biphasic. At [ACh] = 0.1 M both B( *) are released from the AChR . B(2)( *) species. The mechanism involves associative ternary intermediates (AChR . B( *)A, AChR . B( *)A(2) and AChR . B(2)( *)A(2)). The equilibrium constants (K(A)) and dissociation rate constants (k(-A)) for ACh in the ternary complex state R(alpha)' and R(alpha)', respectively, are K(A)' = 1.1 . 10(-2) M and k(-A)' = 3 . 10(5) s(-1) and K(A)' = 7.5 . 10(-2) M and k(-A)' = 2 . 10(6) s(-1). It is of physiological importance that the FCS data indicate that the AChR monomer species (M(r) = 290 000), which normally at [ACh] 1 mM only binds one ACh molecule, does bind two ACh molecules at [ACh] 0.1 M.     

Kinetic analysis of recombinant mammalian α<Subscript>1</Subscript> and α<Subscript>1</Subscript>β glycine receptor channels

To analyze the influence of the beta-subunit on the kinetic properties of GlyR channel currents, alpha(1)-subunits and alpha(1)beta-subunits were transiently expressed in HEK 293 cells. A piezo dimorph was used for fast application of glycine to outside-out patches. The rise time of activation was dose dependent for both receptors and decreased with increasing glycine concentrations. Subunit composition had no effect on the time course of activation. Coexpression of alpha(1)- and beta-subunits resulted in a significantly lower EC(50) and a reduced slope of the dose-response curve of glycine compared with expression of alpha(1)-subunits alone. For both receptor subtypes, the time course of desensitization was concentration dependent. Desensitization was best fitted with a single time constant at 10-30 micro M, with two at 0.1 mM, and at saturating concentrations (0.3-3 mM) with three time constants. Desensitization of homomeric alpha(1)-receptor channels was significantly slower than that of alpha(1)beta-receptor channels. The time course of current decay after the end of glycine pulses was tested at different pulse durations of 1 mM glycine. It was best fitted with two time constants for both alpha(1) and alpha(1)beta GlyR channels, and increased significantly with increasing pulse duration.     

Kinetic characterization of the phencyclidine-N-methyl-D-aspartate receptor interaction: evidence for a steric blockade of the channel

The nature of the interactions between the N-methyl-D-aspartate (NMDA) and the phencyclidine (PCP) receptors was studied in membranes obtained from rat cerebral cortex and washed repeatedly to remove endogenous excitatory amino acids. Binding of [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) to its receptor sites in these membranes proceeded slowly and did not reach equilibrium even after incubation for 4 h at 25 degrees C. The dissociation rate of [3H]TCP-receptor complexes was also slow (t1/2 = 128-165 min). Both association and dissociation followed first-order reaction kinetics, with similar time constants (0.0054 min-1). Addition of glutamate and glycine to the washed membranes was immediately followed by a marked increase in the rates of both association of [3H]TCP with the receptors and its dissociation from them (t1/2 = 8 min). Association now followed second-order reaction kinetics. Accelerated association of [3H]TCP with its binding sites could also be induced by NMDA or by glutamate alone, and glycine enhanced the effect. All effects of glutamate and glycine on [3H]TCP binding kinetics were blocked by the competitive NMDA receptor antagonist AP-5 [D-(-)-2-amino-5-phosphovaleric acid]. [3H]TCP-receptor interactions at equilibrium were not altered by AP-5 or by glutamate and glycine. The binding data were fitted to a model in which interactions of [3H]TCP with the receptor involve a two-step process: the outside ligand must cross a barrier (presumably a closed NMDA receptor channel in the absence of agonists). Once agonists are added, this limitation is removed (presumably because the channel is open).(ABSTRACT TRUNCATED AT 250 WORDS)     

Point mutation in the first transmembrane region of the beta 2 subunit of the gamma--aminobutyric acid type A receptor alters desensitization kinetics of gamma--aminobutyric acid- and anesthetic-induced channel gating

A conserved glycine residue in the first transmembrane (TM1) domain of the beta2 subunit has been identified to be involved with desensitization induced by gamma-aminobutyric acid (GABA) and anesthetics. Recombinant GABA(A) receptors expressed in Sf9 cells were recorded using semi-fast agonist application. Upon direct activation by GABA or anesthetics, the main effect of the TM1 point mutation on the beta2 subunit (G219F) was to slow the time constant (tau) of desensitization. At GABA concentrations eliciting maximum currents, the corresponding median tau values were 0.87 s (25-75% interval (0.76; 1.04 s)), 0.93 s (0.76; 1.23 s), and 1.36 s (1.17; 1.57 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than alpha1beta2gamma2 (p < 0.01) and alpha1(G223F)beta2gamma2 (p < 0.05). For pentobarbital-induced currents (500 microm), the corresponding median tau values were 1.36 s (0.81; 1.41 s), 1.47 s (1.31; 2.38 s), and 2.82 s (2.21; 5.56 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than that for alpha1beta2gamma2 (p < 0.01). The present findings suggest that this TM1 glycine residue is critical for the rate at which desensitization occurs and that both GABA and intravenous anesthetics implement an analogous pathway for generating desensitization.     

Characterization of the gating conformational changes in the felbamate binding site in NMDA channels

The anticonvulsant effect of felbamate (FBM) is ascribable to inhibition of N-methyl-d-aspartate (NMDA) currents. Using electrophysiological studies in rat hippocampal neurons to examine the kinetics of FBM binding to and unbinding from the NMDA channel, we show that FBM modifies NMDA channel gating via a one-to-one binding stoichiometry and has quantitatively the same enhancement effect on NMDA and glycine binding to the NMDA channel. Moreover, the binding rates of FBM to the closed and the open/desensitized NMDA channels are 187.5 and 4.6 x 10(4) M(-1) s(-1), respectively. The unbinding rates of FBM from the closed and the open/desensitized NMDA channels are approximately 6.2 x 10(-2) and approximately 3.1 s(-1), respectively. From the binding and unbinding rate constants, apparent dissociation constants of approximately 300 and approximately 70 microM could be calculated for FBM binding to the closed and the open/desensitized NMDA channels, respectively. The slight (approximately fourfold) difference in FBM binding affinity to the closed and to the open/desensitized NMDA channels thus is composed of much larger differences in the binding and unbinding kinetics (approximately 250- and approximately 60-fold difference, respectively). These findings suggest that the effects of NMDA and glycine binding coalesce or are interrelated before or at the actual activation gate, and FBM binding seems to modulate NMDA channel gating at or after this coalescing point. Moreover, the entrance zone of the FBM binding site very likely undergoes a much larger conformational change along the gating process than that in the binding region(s) of the binding site. In other words, the FBM binding site becomes much more accessible to FBM with NMDA channel activation, although the spatial configurations of the binding ligand(s) for FBM themselves are not altered so much along the gating process.     

CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).     

Receptor occupancy and channel-opening kinetics: a study of GLUR1 L497Y AMPA receptor

AMPA glutamate ion channels are tetrameric receptors in which activation to form the open channel depends on the binding of possibly multiple glutamate molecules. However, it is unclear whether AMPA receptors bound with a different number of glutamate molecules (i.e. one being the minimal and four being the maximal number of glutamate molecules) open the channels with different kinetic constants. Using a laser pulse photolysis technique that provides microsecond time resolution, we investigated the channel-opening kinetic mechanism of a nondesensitizing AMPA receptor, i.e. GluR1Q(flip) L497Y or a leucine-to-tyrosine substitution mutant, in the entire range of glutamate concentrations to ensure receptor saturation. We found that the minimal number of glutamate molecules required to bind to the receptor and to open the channel is two (or n = 2), and that the entire channel-opening kinetics can be adequately described by just one channel-opening rate constant, k(op), which correlates to n = 2. This result suggests that higher receptor occupancy (n = 3 and 4) does not give rise to different k(op) values or, at least, not appreciably if the k(op) values are different. Furthermore, compared with the wild-type receptor (Li, G., and Niu, L. (2004) J. Biol. Chem. 279, 3990-3997), the channel-opening and channel-closing rate constants of the mutant are 1.5- and 13-fold smaller, respectively. Thus, the major effect of this mutation is to decrease the channel-closing rate constant by stabilizing the open channel conformation.     

Kinetic and thermodynamic studies of tet repressor-tetracycline interaction

Stopped-flow measurements have been employed to study the kinetics of the conformational changes in TetR (B) induced by tetracycline binding with and without Mg(2+) ions. Result of stopped-flow fluorometry measurements at pH 8.0 indicate conformational changes in the helix-turn-helix motif in the N-terminal domain and in the C-terminal inducer binding domain. Binding of tetracycline (Tc) to TetR in the absence of Mg(2+) can be described by a simple kinetics process, which is limited to the first step association without any unimolecular conformational change step upon Tc binding. The rate constants for this process are equal to 2.0 x 10(5) M(-)(1) s(-)(1) and 2.1 s(-)(1) for the forward and backward reaction, respectively, and gave the binding constant K(a) = 0.96 x 10(5) M(-)(1). The kinetics of [Tc-Mg](+) binding to TetR can be described by reactions in which the first step describes the association characterized by the rate constants k(a) = 1.4 x 10(5) M(-)(1) s(-)(1) and k(d) = 2.2 x 10(-)(2) s(-)(1) and binding constant K(a) = 6.3 x 10(6) M(-)(1). The first step of [Tc-Mg](+) association is followed by at least three conformational change steps, which occur in the inducer binding site and then propagate to the surroundings of Trp75 and Trp43 residues. The rate constants for the forward, k(c), and backward, k(-)(c), reaction for each of these conformational steps have been determined. The thermodynamics of the binding of tetracycline with and without Mg(2+) to TetR was investigated by isothermal titration calorimetry (ITC) at pH 8.0 and 25 degrees C. The measurement shows that TetR dimer possesses two equivalent binding sites for tetracycline, characterized by binding constant K(a) = 9.0 x 10(6) M(-)(1) and K(a) = 7.0 x 10(4) M(-)(1) for Tc with and without Mg(2+), respectively. The binding of the inducer to TetR, in the presence and absence of Mg(2+) ion, is an enthalpy-driven reaction characterized by DeltaH = -51 kJ mol(-)(1) and DeltaH = -33 kJ mol(-)(1), respectively. The entropy change, DeltaS, for the interaction in the presence of Mg(2+) is equal to -38.9 J K(-)(1) mol(-)(1), and for the tetracycline alone, it was estimated at -17.6 J K(-)(1) mol(-)(1).     

Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.     

Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line

Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.     

Estrogen receptor in chicken oviduct: receptor dissociation kinetics and transformation

Cytosolic and nuclear estrogen receptor forms of chicken oviduct have been studied by (1) measuring hormone dissociation kinetics and by (2) sucrose density gradient analysis on high salt gradients. Estradiol dissociates from the receptor in chicken oviduct cytosol at 22 degrees C following a two-phase exponential process. The fraction of receptor with a fast dissociation rate (k = 120 X 10(-3) min-1) decreases as a function of the pre-incubation at 22 degrees C; after prolonged pre-incubation only the slowly dissociating (k = 12.3 X 10(-3) min-1) form remains. Dissociation of moxestrol, a synthetic estrogen with a higher affinity, from the cytosol receptor at 30 degrees C is similar, showing a transition of a fast dissociating form (k = 120 X 10(-3) min-1) to a slowly dissociating form (k = 7.6 X 10(-3) min-1) as a result of pre-incubation at 30 degrees C. A concomitant temperature-dependent shift of the estrogen receptor from a 4.8 S to a 6.1 S form was observed with moxestrol but not with estradiol as a ligand. Sodium molybdate (20 mM) and NaSCN (400 mM) inhibit the temperature-dependent increase in sedimentation coefficient, but molybdate allows the formation of a receptor form which shows intermediary dissociation kinetics. Estrogen receptor, precipitated with ammonium sulfate (0-35%) shows monophasic dissociation kinetics of estradiol (k = 39.5 X 10(-3) min-1) and for moxestrol (k = 10.8 X 10(-3) min-1), suggesting full receptor activation only with moxestrol as a ligand. Moxestrol-receptor complexes obtained by ammonium sulfate precipitation sediment at 0 degree C at 4.8 S. Only after subsequent incubation at 30 degrees C a shift from 4.8 S to 5.9 S is observed, suggesting that the formation of the slowly dissociating form of the receptor may precede the formation of a stable transformed receptor complex. The nuclear estrogen receptor with estradiol as a ligand shows biphasic dissociation kinetics at 22 degrees C (k = 70 X 10(-3) min-1; k = 14.0 X 10(-3) min-1). The ratio of both components (1:1) does not change after preincubation of the nuclear receptor extract at 22 degrees C. Moxestrol dissociates from the nuclear receptor at 30 degrees C monophasically with a slow rate (k = 6.1 X 10(-3) min-1), suggesting that it is extracted as an activated hormone-receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation and desensitization of the recombinant P2X1 receptor at nanomolar ATP concentrations

Activation and desensitization kinetics of the rat P2X1 receptor at nanomolar ATP concentrations were studied in Xenopus oocytes using two-electrode voltage-clamp recording. The solution exchange system used allowed complete and reproducible solution exchange in <0.5 s. Sustained exposure to 1-100 nM ATP led to a profound desensitization of P2X1 receptors. At steady-state, desensitization could be described by the Hill equation with a K1/2 value of 3.2 +/- 0.1 nM. Also, the ATP dependence of peak currents could be described by a Hill equation with an EC50 value of 0.7 microM. Accordingly, ATP dose-effect relationships of activation and desensitization practically do not overlap. Recovery from desensitization could be described by a monoexponential function with the time-constant tau = 11.6 +/-1.0 min. Current transients at 10-100 nM ATP, which elicited 0.1-8.5% of the maximum response, were compatible with a linear three-state model, C-O-D (closed-open-desensitized), with an ATP concentration-dependent activation rate and an ATP concentration-independent (constant) desensitization rate. In the range of 18-300 nM ATP, the total areas under the elicited current transients were equal, suggesting that P2X1 receptor desensitization occurs exclusively via the open conformation. Hence, our results are compatible with a model, according to which P2X1 receptor activation and desensitization follow the same reaction pathway, i.e., without significant C to D transition. We assume that the K1/2 of 3.2 nM for receptor desensitization reflects the nanomolar ATP affinity of the receptor found by others in agonist binding experiments. The high EC50 value of 0.7 microM for receptor activation is a consequence of fast desensitization combined with nonsteady-state conditions during recording of peak currents, which are the basis of the dose-response curve. Our results imply that nanomolar extracellular ATP concentrations can obscure P2X1 receptor responses by driving a significant fraction of the receptor pool into a long-lasting refractory closed state.     

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2 = 10.7 s(-1)) and the iso step (k5 = 12.1 s(-1)). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4 = 3.9 s(-1)) and the iso step (k5 = 12.1 s(-1)), while the chemical step is fast (k2 = 32.3 s(-1)). The transient kinetic studies are generally consistent with the steady state kcat and Km values. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained from k(-1)/k1, are 3.5 nM for 8-oxo-dGTP and 62 microM for dGTP, indicating that 8-oxo-dGTP binds 1.8 x 10(4)-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.     

Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme.

The kinetics of protein fluorescence change exhibited by ADP or orthophosphate addition to the Mg2+-or Mn2+-activated unadenylylated glutamine synthetase from Escherichia coli were studied. The kinetic patterns of these reactions are incompatible with a simple bimolecular binding process and a mechanism which required protein isomerization prior to substrate binding. They are consistent with a mechanism in which direct substrate binding is followed by a substrate-induced conformational change step, ES in equilibrium ES. At pH 7.0 and 15 degrees C, the association constants for the direct binding (K1) of ADP to MnE1.0 and of Pi to MnE1.0ADP are 3.9 X 10(4) and 2.28 X 10(2) M(-1), respectively. The association constant for the direct binding of ADP to MnE1.0Pi is 2.3 X 10(4) M(-1) at pH 7.0 and 19 degrees C. The deltaG degrees for the substrate-induced conformational step are -3.5 and -1.3 kcal mol(-1) due to ADP binding to MnE1.0Pi and MnE1.0, respectively, and -1.4 kcal mol(-1) due to Pi binding to MnE1.0ADP. Rate constants, k2 and k(-2), for the isomerization step are: 90 and 9.5 s(-1) for ADP binding to MnE1.0, 440 and 0.36 s(-1) for ADP binding to MnE1.0Pi, and 216 and 1.8 s(-1) for Pi binding to MnE1.0ADP. Due to low substrate affinity, the association constant for direct Pi binding to MnE1.0 was roughly estimated to be 230 M(-1) and k2 = 750 s(-1), k(-2) = 250 s(-1). At 9 degrees C and pH 7.0, the estimated association constants for the direct ADP binding to MgE1.0 and MgE1.0 Pi are 1.8 X 10(4) and 1.6 X 10(4) M(-1), respectively; and the rate constants for the isomerization step associated with the corresponding reaction are k2 = 550 s(-1), k(-2) = 500 s(-1), and k2 = 210 s(-1), k(-2) = 100 s(-1). From the kinetic analysis it is evident that the inability of Mn2+ to support biosynthetic activity of the unadenylylated enzyme is due to the slow rate of ADP release from the MnE1.0PiADP complex. In contrast the large k(-2) obtained for ADP release from the MgE1.0ADP or MgE1.0PiADP complex indicates that this step is not rate limiting in the biosynthesis of glutamine since the k catalysis obtained under the same conditions is 7.2 s(-1).     

Inhibition of ionotropic neurotransmitter receptors by antagonists: strategy to estimate the association and the dissociation rate constant of antagonists with very strong affinity to the receptors.

Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)