Opening Rate Constant Research Articles

Gating of acetylcholine receptor channels: brownian motion across a broad transition state.

Acetylcholine receptor channels (AChRs) are proteins that switch between stable "closed" and "open" conformations. In patch clamp recordings, diliganded AChR gating appears to be a simple, two-state reaction. However, mutagenesis studies indicate that during gating dozens of residues across the protein move asynchronously and are organized into rigid body gating domains ("blocks"). Moreover, there is an upper limit to the apparent channel opening rate constant. These observations suggest that the gating reaction has a broad, corrugated transition state region, with the maximum opening rate reflecting, in part, the mean first-passage time across this ensemble. Simulations reveal that a flat, isotropic energy profile for the transition state can account for many of the essential features of AChR gating. With this mechanism, concerted, local structural transitions that occur on the broad transition state ensemble give rise to fractional measures of reaction progress (Phi values) determined by rate-equilibrium free energy relationship analysis. The results suggest that the coarse-grained AChR gating conformational change propagates through the protein with dynamics that are governed by the Brownian motion of individual gating blocks.

Structural Effects on the β-Scission Reaction of Alkoxyl Radicals. Direct Measurement of the Absolute Rate Constants for Ring Opening of Benzocycloalken-1-oxyl Radicals

[reaction: see text] The absolute rate constants for beta-scission of a series of benzocycloalken-1-oxyl radicals and of the 2-(4-methylphenyl)-2-butoxyl radical have been measured directly by laser flash photolysis. The benzocycloalken-1-oxyl radicals undergo ring opening with rates which parallel the ring strain of the corresponding cycloalkanes. In the 1-X-indan-1-oxyl radical series, ring opening is observed when X = H, Me, whereas exclusive C-X bond cleavage occurs when X = Et. The factors governing the fragmentation regioselectivity are discussed.

Activation of GABA(A) receptors containing the alpha4 subunit by GABA and pentobarbital.

The activation properties of GABA(A) receptors containing alpha4beta2gamma2 and alpha4beta2delta subunits were examined in the presence of GABA or pentobarbital. The receptors were expressed transiently in HEK 293 cells, and the electrophysiological experiments were carried out using cell-attached single-channel patch clamp or whole-cell macroscopic recordings. The data show that GABA is a stronger activator of alpha4beta2gamma2 receptors than alpha4beta2delta receptors. Single-channel clusters were recorded from alpha4beta2gamma2 receptors in the presence of 10-5000 microm GABA. The maximal intracluster open probability was 0.35, with a half-maximal response elicited by 32 microm GABA. Simultaneous kinetic analysis of single-channel currents obtained at various GABA concentrations yields a channel opening rate constant of 250 s(-1), and a K(D) of 20 microm. In contrast, only isolated openings were observed in the presence of GABA for the alpha4beta2delta receptor. Pentobarbital was a strong activator of both alpha4beta2gamma2 and alpha4beta2delta receptors. The maximal cluster open probability, recorded in the presence of 100 microm pentobarbital, was 0.7. At higher pentobarbital concentrations, the cluster open probability was reduced, probably due to channel block. The results from single-channel experiments were confirmed by macroscopic recordings from HEK cells in the presence of GABA or pentobarbital.

Life at the top: the transition state of AChR gating.

Most neurotransmitter receptors belong to either the pentameric nicotinoid receptor family or the tetrameric glutamatergic receptor family. The muscle nicotinic acetylcholine receptor (AChR), the prototype of the nicotinoid receptor family, gates by switching between a closed configuration (in which ion permeation is forbidden) and an open configuration (which allows ions to pass through). Rate-equilibrium linear free energy relationship analysis has allowed us to explore the transition state that links these two stable conformations. A series of point mutations were made to individual AChR residues, and the ensuing changes in the rate constants of channel opening and closing for the fully liganded receptor were determined. These experiments suggest that gating occurs approximately as a reversible, solitary conformational wave that propagates between the neurotransmitter binding site and the membrane domain, along the long axis of the receptor. A detailed knowledge of the gating mechanism can serve as a basis for understanding the shape of the postsynaptic ion current and for the differences in synaptic responses among different ligand-gated channels.

Destabilization of the HIV-1 Complementary Sequence of TAR by the Nucleocapsid Protein Through Activation of Conformational Fluctuations

The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two obligatory strand transfer reactions required for the synthesis of a complete proviral DNA by reverse transcriptase. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response region (TAR) sequence at the 3′ end of the genomic RNA and the complementary sequence cTAR at the 3′ terminus of minus strong-stop DNA, the early product of reverse transcription. In order to determine the dynamics of NCp7-mediated nucleic acid destabilization, we investigated by time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly labeled cTAR sequence with NC(12–55) containing NCp7 CCHC zinc fingers and flanking basic amino acid residues. From the chemical rates and the activation energy associated with the conformational fluctuations observed in the absence of NC, it is concluded that such fluctuations are associated with the opening and closing of the double-stranded terminal segments of cTAR. The destabilizing activity of NC(12–55) occurs mainly through a major increase of the opening rate constant of cTAR. Moreover, NC appears to augment the number of pathways between the open and closed states of cTAR, suggesting that it initiates melting of base-pairs at different locations within the terminal segments of cTAR. This activity of NC on the dynamics of cTAR secondary structure is thought to be critical for the formation of the cTAR–TAR complex, which is essential for the specificity and extent of proviral DNA synthesis by reverse transcriptase.

Low doses of ethanol and a neuroactive steroid positively interact to modulate rat GABA(A) receptor function.

Fast inhibitory responses in the central nervous system are mediated by the GABA(A) receptor. The activation and function of the GABA(A) receptor can be modulated by a variety of compounds including benzodiazepines, barbiturates and neuroactive steroids. Modulation of the GABA(A) receptor function by ethanol has been observed in some but not all studies. We have studied the effect of ethanol at concentrations corresponding to light intoxication on the function of the recombinant GABA(A) receptor containing alpha1beta2gamma2 subunits. The experiments were performed both in the absence and presence of low, subthreshold concentrations of a neuroactive steroid. The results demonstrate that, in the presence of the steroid, 0.05 % (9 mM) ethanol potentiates the GABA(A) receptor function by increasing the channel mean open duration. No effect was observed on the channel closed time durations. The data suggest that ethanol influences channel closing with no effect on the affinity of the receptor for GABA or the channel opening rate constant.

Contributions of the non-alpha subunit residues (loop D) to agonist binding and channel gating in the muscle nicotinic acetylcholine receptor.

The agonist binding site of the nicotinic acetylcholine receptor has a loop-based structure, and is formed by residues located remotely to each other in terms of primary structure. Amino acid residues in sites delta57 and delta59, and the equivalent residues in the epsilon; subunit, have been identified as part of the agonist binding site and designated as loop D. The effects of point mutations in sites delta57, delta59, epsilon;55 and epsilon;57 on agonist binding and channel gating were studied. The mutated receptors were expressed transiently in HEK 293 cells and the currents were recorded using the cell-attached single-channel patch clamp technique. The results demonstrate that the mutations mainly affect channel gating with the major portion of the effect due to a reduction in the channel opening rate constant. For both the delta57/epsilon;55 and the delta59/epsilon;57 site, a mutation in the epsilon; subunit had a greater effect on channel gating than a mutation in the delta subunit. In all instances, agonist binding was affected to a lesser degree than channel gating. Previous data have placed the delta57 and delta59 residues in or near the agonist binding pocket. The data presented here suggest that these two residues (and the homologous sites in the epsilon; subunit) are not involved in specific interactions with the nicotinic agonist and that they affect the activation of the nicotinic receptor by shaping the overall structure of the agonist binding site.

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

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

PH and urea dependence of amide hydrogen-deuterium exchange rates in the beta-trefoil protein hisactophilin.

Amide hydrogen/deuterium exchange rates were measured as a function of pH and urea for 37 slowly exchanging amides in the beta-trefoil protein hisactophilin. The rank order of exchange rates is generally maintained under different solution conditions, and trends in the pH and urea dependence of exchange rates are correlated with the rank order of exchange rates. The observed trends are consistent with the expected behavior for exchange of different amides via global and/or local unfolding. Analysis of the pH dependence of exchange in terms of rate constants for structural opening and closing reveals a wide range of rates in different parts of the hisactophilin structure. The slowest exchanging amides have the slowest opening and closing rates. Many of the slowest exchanging amides are located in trefoil 2, but there are also some slow exchanging amides in trefoils 1 and 3. Slow exchangers tend to be near the interface between the beta-barrel and the beta-hairpin triplet portions of this single-domain structure. The pattern of exchange behaviour in hisactophilin is similar to that observed previously in interleukin-1 beta, indicating that exchange properties may be conserved among beta-trefoil proteins. Comparisons of opening and closing rates in hisactophilin with rates obtained for other proteins reveal clear trends for opening rates; however, trends in closing rates are less apparent, perhaps due to inaccuracies in the values used for intrinsic exchange rates in the data fitting. On the basis of the pH and urea dependence of exchange rates and optical measurements of stability and folding, EX2 is the main exchange mechanism in hisactophilin, but there is also evidence for varying levels of EX1 exchange at low and high pH and high urea concentrations. Equilibrium intermediates in which subglobal portions of structure are cooperatively disrupted are not apparent from analysis of the urea dependence of exchange rates. There is, however, a strong correlation between the Gibbs free energy of opening and the denaturant dependence of opening for all amides, which suggests exchange from a continuum of states with different levels of structure. Intermediates are not very prominent either in equilibrium exchange experiments or in quenched-flow kinetic studies; hence, hisactophilin may not form partially folded states as readily as IL-1 beta and other beta-trefoil proteins.

Aromatics at the murine nicotinic receptor agonist binding site: mutational analysis of the alphaY93 and alphaW149 residues.

1. Two aromatic residues of the muscle nicotinic receptor putative agonist binding site, a tyrosine in position alpha93 and a tryptophan in position alpha149, were mutated to phenylalanine and the effects of the mutations on receptor properties were investigated using single-channel patch clamp. 2. The alphaY93F mutation reduced the receptor affinity by approximately 4-fold and the channel opening rate constant by 48-fold. The alphaW149F mutation reduced the receptor affinity by approximately 12-fold and the channel opening rate constant by 93-fold. 3. The kinetic properties of hybrid receptors that contained one wild-type and one mutated alpha subunit were also examined. Only one type of hybrid receptor activity was detected. The hybrid receptors had a channel opening rate constant intermediate to those of the wild-type and mutant receptors. It was concluded that the ligand binding sites in the mutated muscle nicotinic receptor contributed equally to channel gating. In the case of the alphaW149F mutation, the presence of the mutation in one of the binding sites had no effect on the binding properties of the other, non-mutated, site. 4. The mutant channel opening and closing rate constants were also estimated in the presence of tetramethylammonium. The data suggested significant interaction between the acetyl group of acetylcholine and the alphaY93 residue.

Surfing the Conformational Wave of Acetylcholine Receptor Channel Gating

The muscle nicotinic acetylcholine receptor channel (AChR) is a cylindrical allosteric membrane protein (∼120 x 60 Å Fig. 1) that adopts alternative quaternary conformations (“open” and “closed”) with different functional properties (ion-conducting and ion-impermeable, respectively). We have characterized, residue-by-residue, the dynamics of the conformational change associated with gating using the framework of linear free energy relationships (LFER). The sequence of molecular events that underlies the closed-to-open gating transition was inferred from kinetic measurements of the receptor at the single molecule level.Specific regions of the AChR were perturbed using site-directed mutagenesis, changes in the membrane potential, or different agonists. Single-channel currents were recorded from cell-attached patches (Fig. 2). For the gain-of-function mutations, choline was used as the agonist because of its low efficacy. The opening rate constant was determined at a saturating concentration of agonist (for choline, 20 mM) in order to isolate gating from binding steps. to avoid bias introduced by fast channel blockade, the closing rate constant was measured at a low concentration (for choline, 200 μM). The diliganded channel opening (β) and closing (α) rate constants were estimated using the QuB suite of kinetic analysis programs. in general, a plot of the log rate constant vs. log equilibrium constant was linear.

Conrotatory photochemical ring opening of alkylcyclobutenes in solution. A test of the hot ground-state mechanism.

Quantum yields for photochemical ring opening of six alkylcyclobutenes have been measured in hexane solution using 228-nm excitation, which selectively populates the lowest pi,R(3s) excited singlet states of these molecules and has been shown previously to lead to ring opening with clean conrotatory stereochemistry. The compounds studied in this work-1,2-dimethylcyclobutene (1), cis- and trans-1,2,3,4-tetramethylcyclobutene (cis- and trans-5), hexamethylcyclobutene (8), and cis- and trans-tricyclo[6.4.0.0(2,7)]dodec-1(2)-ene (cis- and trans-9)-were selected so as to span a broad range in molecular weight and as broad a range as possible in Arrhenius parameters for thermal (ground-state) ring opening. RRKM calculations have been carried out to provide estimates of the rate constants for ground-state ring opening of each of the compounds over a range of thermal energies from 20 000 to 49 000 cm(-1). These have been used to estimate upper limits for the quantum yields of ring opening via a hot ground-state mechanism, assuming a value of k(deact) = 10(11) s(-1) for the rate constant for collisional deactivation by the solvent, that internal conversion to the ground state from the lowest Rydberg state occurs with close to unit efficiency, and that ergodic behavior is followed. The calculated quantum yields are significantly lower than the experimental values in all cases but one (1). This suggests that the Rydberg-derived ring opening of alkylcyclobutenes is a true excited-state process and rules out the hot ground-state mechanism for the reaction.

Highly regioselective anaerobic photocyclization of 3-styrylpyridines.

The photochemical behavior of the cis isomers of the three isomeric styrylpyridines and two (aminostyryl)pyridines has been investigated under aerobic and anaerobic conditions. Both 3-styrylpyridine and its 3'-amino derivative undergo highly regioselective formation of 2-azaphenanthrene products under anaerobic conditions. In the presence of oxygen, mixtures of 4- and 2-azaphenanthrene products are obtained. The formation of 2-azaphenanthrenes in the absence of oxygen is attributed to conversion of the 4a,4b-dihydroazaphenanthrene primary photoproduct to a 1,4-dihydropyridine intermediate by means of a formal 1,7-hydrogen shift. This intermediate is moderately stable in the absence of oxygen and has been characterized by comparison of its (1)H NMR and electronic absorption spectra with calculated spectra. This intermediate is converted to the 2-azaphenanthrene in both the absence and presence of oxygen. The regioselectivity of photocyclization of 3-substituted stilbenes and related diarylethylenes is suggested to depend on the relative rate constants for ring opening and sigmatropic rearrangements of the dihydrophenanthrene intermediates as well as their rates of reaction with oxygen or other oxidants.

Activation and block of recombinant GABA(A) receptors by pentobarbitone: a single-channel study.

Recombinant GABA(A) receptors (alpha1beta2gamma2L) were transiently expressed in HEK 293 cells. We have investigated activation and block of these receptors by pentobarbitone (PB) using cell-attached single-channel patch clamp. Clusters of single-channel activity elicited by 500 microM PB were analysed to estimate rate constants for agonist binding and channel gating. The minimal model able to describe the kinetic data involved two sequential binding steps, followed by channel opening. The estimated channel opening rate constant is approximately 1500 s(-1), and the estimated equilibrium dissociation constants for the binding steps involved in activation are approximately 2 mM. Our results show a dose-dependent block of receptors at millimolar concentrations of PB that results in reduced open interval durations. The reduction in mean open time is linearly proportional to PB concentration, indicating that block can be produced by binding of a single PB molecule. Addition of millimolar concentrations of PB in the presence of GABA also produces a reduction of open channel lifetime in addition to a progressive increase in the closed interval durations within a cluster. The data suggest that the receptor contains two or more blocking sites while occupancy of only one of the sites is sufficient for channel block. Neither the blocking rate constant nor return rate from the blocked state(s) is affected by pH (ionization status of the PB molecule) demonstrating that both neutral and anionic forms of PB cause channel block.

Voltage dependence of the glycine receptor-channel kinetics in the zebrafish hindbrain.

Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic currents (mIPSCs), depolarizing the patch produced a broadening of the transient outside-out current evoked by short applications (1 ms) of a saturating concentration of glycine (3 mM). When the outside-out patch was depolarized from -50 to +20 mV, the peak current varied linearly with voltage. A 1-ms application of 3 mM glycine evoked currents that activated rapidly and deactivated biexponentially with time constants of approximately 5 and approximately 30 ms (holding potential of -50 mV). These two decay time constants were increased by depolarization. The fast deactivation time constant increased e-fold per 95 mV. The relative amplitude of the two decay components did not significantly vary with voltage. The fast component represented 64.2 +/- 2.8% of the total current at -50 mV and 54.1 +/- 10% at +20 mV. The 20-80% rise time of these responses did not show any voltage dependence, suggesting that the opening rate constant is insensitive to voltage. The 20-80% rise time was 0.2 ms at -70 mV and 0.22 ms at +20 mV. Responses evoked by 100-200 ms application of a low concentration of glycine (0.1 mM) had a biphasic rising phase reflecting the complex gating behavior of the glycine receptor. The time constant of these two components and their relative amplitude did not change with voltage, suggesting that modal shifts in the glycine-activated channel gating mode are not sensitive to the membrane potential. Using a Markov model to simulate glycine receptor gating behavior, we were able to mimic the voltage-dependent change in the deactivation time course of the responses evoked by 1-ms application of 3 mM glycine. This kinetics model incorporates voltage-dependent closing rate constants. It provides a good description of the time course of the onset of responses evoked by the application of a low concentration of glycine at all membrane potentials tested.

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.

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.

Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels.

The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.

Channel-opening mechanism of a kainate-activated glutamate receptor: kinetic investigations using a laser-pulse photolysis technique.

Kainate is an excitatory neurotransmitter that binds to the kainate and AMPA receptor subtypes of the glutamate receptor and triggers the formation of cation permeable transmembrane channels in these receptors. In the present report the channel-opening mechanism of the AMPA receptors by kainate has been determined in rat hippocampal neurons using two different kinetic methods, namely, the rapid-flow method (cell-flow) with a 10 ms time resolution and a laser-pulse photolysis technique with a approximately 65 microseconds time resolution. The whole-cell currents induced by kainate, using the cell-flow method, are nondesensitizing and inhibited significantly by CNQX and hence pertain to activation of the AMPA receptors and not the kainate receptors. The cell-flow measurements were used to evaluate the constants pertaining to the minimum mechanism that could account for the concentration of the receptor in the open-channel form over a 500-fold range of kainate concentration. These constants, namely, the intrinsic dissociation constant of kainate from the AMPA receptor and the channel-opening equilibrium constant, were determined to be 140 +/- 30 microM and 8 +/- 2, respectively. On the other hand, the kinetics of the steps leading to channel opening was evaluated using the laser-pulse photolysis techniques. In this technique whole-cell currents were obtained by releasing kainate in the submillisecond time scale near the cell by photolysis of N-(alpha-carboxy-2-nitrobenzyl) kainate. The concentration of the released kainate was calculated by comparing the whole-cell currents obtained from the laser-pulse photolysis experiments with the whole currents obtained with 100 microM kainate on the same cell using cell-flow measurements. The rate constants for channel opening and closing were then determined from the observed rate constants for the current rise obtained as a function of kainate concentration. These rates were 5000 +/- 2000 and 640 +/- 30 s-1, respectively. The rate and equilibrium constants obtained in the present report allow an evaluation of the fraction of the receptors in the open-channel form as a function of time and kainate concentration, hence providing insight into the role of kainate in neuronal signal transmission.

Depolarization Increases the Single-Channel Conductance and the Open Probability of Crayfish Glutamate Channels

We have studied the voltage sensitivity of glutamate receptors in outside-out patches taken from crayfish muscles. We found that single-channel conductance, measured directly at the single-channel level, increases as depolarization rises. At holding potentials from −90 mV to ∼20 mV, the conductance is 109 pS. At holding potentials positive to 20 mV, the conductance is 213 pS. This increase in single-channel conductance was also observed in cell-attached patches. In addition, desensitization, rise time, and the dose-response curve were all affected by depolarization. To further clarify these multifaceted effects, we evaluated the kinetic properties of single-channel activity recorded from cell-attached patches in hyperpolarization (membrane potential around −75 mV) and depolarization (membrane potential ∼105 mV). We found that the glutamate dissociation rate constant ( k −) was affected most significantly by membrane potential; it declined 6.5-fold under depolarization. The rate constant of channel closing ( k c) was also significantly affected; it declined 1.8-fold. The rate constant of channel opening ( k o) declined only 1.2-fold. The possible physiological significance of the depolarization-mediated changes in the above rate constants is discussed.