Channel Opening Rate Constant Research Articles

Activation and Modulation of Concatemeric GABA-A Receptors Expressed in Human Embryonic Kidney Cells

We have employed whole-cell and single-channel electrophysiology to examine the kinetic and pharmacological properties of GABA-A receptors consisting of gamma2L-beta2-alpha1 and beta2-alpha1 subunit concatemeric constructs expressed in human embryonic kidney cells. Concatemeric receptors activated by GABA exhibited the same single-channel conductance, channel opening rate constant, and basic open- and closed-time properties as receptors containing free subunits. However, the whole-cell GABA dose-response and the single-channel effective opening rate curves were shifted to higher GABA concentrations, suggesting that the concatemeric receptors have a lower affinity to GABA. Pharmacological tests demonstrated that the concatemeric receptors were potentiated by pentobarbital, diazepam, and the neurosteroid (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP), and were insensitive to Zn(2+). Selective introduction of the alpha1Q241L mutation, previously shown to abolish alpha1beta2gamma2L channel potentiation by neurosteroids, into one of the two concatemeric constructs had a relatively small effect on receptor activation by GABA or macroscopic potentiation by the neurosteroid 3alpha5alphaP. Single-channel measurements showed that the kinetic mechanism of action of the steroid is unchanged when the mutation is introduced to the gamma2L-beta2-alpha1 concatemer. We infer that a single wild-type alpha subunit is capable of mediating the full set of kinetic effects in the presence of steroids. Introduction of the alpha1Q241W mutation, previously shown to mimic the effect of the steroid on alpha1beta2gamma2L channels, selectively into either concatemeric construct altered the mode of activity elicited by P4S, but the presence of mutations in both alpha subunits was required to affect open-time distributions. The data indicate that the alpha1Q241W mutation acts as a partial steroid modulator.

Aromatic Residues ∈Trp-55 and δTrp-57 and the Activation of Acetylcholine Receptor Channels

The two transmitter binding sites of the neuromuscular acetylcholine (ACh) receptor channel contain several aromatic residues, including a tryptophan located on the complementary, negative face of each binding pocket. These two residues, Trp-55 in the epsilon subunit and Trp-57 in the delta subunit, were mutated (AEFHILRVY), and for most constructs the rate constants for acetylcholine binding and channel gating were estimated by using single channel kinetic analyses. The rate constants for unliganded channel opening and closing were also estimated for some mutants. From these measurements we calculated all of the equilibrium constants of the "allosteric" cycle as follows: diliganded gating, unliganded gating, dissociation from the C(losed) conformation, and dissociation from the O(pen) conformation. The results indicate the following. (i) These aromatic side chains play a relatively minor role in ACh receptor channel activation. (ii) The main consequence of mutations is to reduce the affinity of the O conformation of the binding site for ACh, with the effect being greater at the epsilon subunit. (iii) In epsilon (but not delta) the aromatic nature of the side chain is important in determining affinity, to a slightly greater degree in the O conformation. Phi value analyses (of both tryptophan residues) show Phi approximately 1 for both the ACh binding and diliganded gating reactions. (iv) This suggests that the structural boundaries of the dynamic elements of the gating conformational change may not be subunit-delimited, and (v) the mutated tryptophan residues experience energy changes that occur relatively early in both the ligand-binding and channel-gating reactions.

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.

GluR3 Flip and Flop: Differences in Channel Opening Kinetics

Ample evidence from earlier studies of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, GluR3 included, suggests that alternative splicing not only enriches AMPA receptor diversity but also, more importantly, creates receptor variants that are functionally different. However, it is not known whether alternative splicing affects the receptor channel opening that occurs in the microsecond time domain. Using a laser-pulse photolysis technique combined with whole-cell recording, we characterized the channel opening rate process for two alternatively spliced variants of GluR3, i.e., GluR3flip and GluR3flop. We show that the alternative splicing that generates flip and flop variants of GluR3 receptors regulates the channel opening process by controlling the rate of channel closing but not the rate of channel opening or the glutamate binding affinity. Specifically, the flop variant closes its channel almost 4-fold faster than the flip variant. We therefore propose that the function of the flip-flop sequence module in the channel opening process of AMPA receptors is to stabilize the open channel conformation, presumably by its pivotal structural location. Furthermore, a comparison of the flip isoform among all AMPA receptor subunits, based on the magnitude of the channel opening rate constant, suggests that GluR3 is kinetically more similar to GluR2 and GluR4 than to GluR1.

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.

A speed limit for conformational change of an allosteric membrane protein

Neuromuscular acetylcholine receptors are synaptic ion channels that open and close with rate constants of approximately equal to 48,000 s(-1) and approximately equal to 1,700 s(-1), respectively (in adult mouse, at 24 degrees C, -100 mV membrane potential). Perturbations of many different sites in the protein can change these rate constants, with those in the extracellular domain mainly affecting channel-opening and many of those in the membrane and intracellular domains mainly affecting channel-closing. We used single-channel recordings to measure the total open time per activation (tau(b)) elicited by a low concentration of the natural transmitter, acetylcholine. tau(b) increased in constructs with mutations that increased the gating equilibrium constant by either increasing the opening or decreasing the closing rate constant. However, tau(b) did not approach the same asymptote in fast-opening and slow-closing constructs. The maximum value for the slow closers was about twice that for the fast openers. One interpretation of this difference is that there is an upper limit to the channel-opening rate constant, which we estimate to be approximately 0.86 mus(-1). One possibility is that this limit is the rate of conformational change in the absence of an overall activation barrier and thus reflects the kinetic prefactor for the acetylcholine receptor opening isomerization.

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.

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.

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.

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.

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.

Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel.

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast-gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)-liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.

A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration-jump techniques.

1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside-out and whole-cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside-out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s-1, and the closing rate constant is 29 s-1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11-15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside-out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside-out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside-out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600.

Neurotransmission parameters estimated from miniature endplate current growth phase

A numerical model of miniature endplate current (mepc) generation was fitted to the rising phase of individual mepcs recorded at the frog neuromuscular junction, and estimates of 6 transmission parameters were obtained. Model fitting was enabled by assuming literature values for geometric parameters and determining single channel current by noise analysis, the channel closing rate constant from the mepc decay, and acetylcholine hydrolysis parameters from mepcs recorded in esterase-blocked endplates. Under control conditions, mean estimates were: number of molecules in a quantum = 29,000, diffusion coefficient = 2.8 × 10 −6cm 2s −1, endplate receptor density = 8500 μm −2, forward binding rate constant = 7.6 × 10 8 M −1s −1, equilibrium dissociation constant = 58 μM and channel opening rate constant = 8100 s −1.

Acetylcholine-induced current in perfused rat myoballs.

Spherical "myoballs" were grown under tissue culture conditions from striated muscle of neonatal rat thighs. The myoballs were examined electrophysiologically with a suction pipette which was used to pass current and perfuse internally. A microelectrode was used to record membrane potential. Experiments were performed with approximately symmetrical (intracellular and extracellular) sodium aspartate solutions. The resting potential, acetylcholine (ACh) reversal potential, and sodium channel reversal potential were all approximately 0 mV. ACh-induced currents were examined by use of both voltage jumps and voltage ramps in the presence of iontophoretically applied agonist. The voltage-jump relaxations had a single exponential time-course. The time constant, tau, was exponentially related to membrane potential, increasing e-fold for 81 mV hyperpolarization. The equilibrium current-voltage relationship was also approximately exponential, from -120 to +81 mV, increasing e-fold for 104 mV hyperpolarization. The data are consistent with a first-order gating process in which the channel opening rate constant is slightly voltage dependent. The instantaneous current-voltage relationship was sublinear in the hyperpolarizing direction. Several models are discussed which can account for the nonlinearity. Evidence is presented that the "selectivity filter" for the ACh channel is located near the intracellular membrane surface.