Photoactivation and dissociation of agonist molecules at the nicotinic acetylcholine receptor in voltage-clamped rat myoballs.

The photochemical properties of the azobenzene derivative, Bis-Q, were exploited to carry out an agonist concentration jump followed by a molecular rearrangement of bound agonist molecules at acetylcholine (ACh) receptor channels of voltage-clamped rat myoballs. Myoballs were bathed in solutions containing low concentrations of cis-Bis-Q, the inactive isomer. Whole-cell current relaxations were studied following a light flash that produced a concentration jump of agonist, trans-Bis-Q, followed by a second flash that produced net trans----cis photoisomerizations of Bis-Q molecules. The concentration-jump relaxation provided a measure of the mean burst duration for ACh receptor channels occupied by trans-Bis-Q (7.7 ms, 22 degrees C). The second current relaxation was a more rapid conductance decrease (phase 1, tau = 0.8 ms). Phase 1 may represent either the burst duration for receptors initially occupied by a single cis- and a single trans-Bis-Q molecule or that for unliganded receptors. Single-channel current recordings from excised outside-out membrane patches showed that single channels open following an agonist concentration jump comparable to that used in the whole-cell experiments; when many such records were averaged, a synthetic macroscopic relaxation was produced. Individual open channels closed faster following a flash that promoted trans----cis photoisomerizations of the bound ligand, thus confirming the whole-cell observations of phase 1.     

Response of acetylcholine receptors to photoisomerizations of bound agonist molecules.

In these experiments, agonist-induced conductance is measured while a sudden perturbation is produced at the agonist-receptor binding site. A voltage-clamped Electrophorus electroplaque is exposed to trans-Bis-Q, a potent agonist. Some channels are open; these receptors have bound agonist molecules. A light flash isomerizes 3(-35)% of the trans-Bis-Q molecules to their cis form, a far poorer agonist. This causes a rapid decrease of membrane conductance (phase 1), followed by a slower increase (phase 2). Phase 1 has the amplitude and wavelength dependence expected if the channel closes within 100 mus after a single bound trans-Bis-Q is isomerized, and if the photochemistry of bound Bis-Q resembles that in solution. Therefore, the receptor channel responds rapidly, and with a hundred-fold greater closing rate, after this change in the structure of a bound ligand. Phase 2 (the conductance increase) seems to represent the relaxation back toward equilibrium after phase 1, because (a) phase 2 has the same time constant (1(-5) ms) as a voltage- or concentration-jump relaxation under identical conditions; and (b) phase 2 is smaller if the flash has led to a net decrease in (trans-Bis-Q). Still slower signals follow: phase 3, a decrease of conductance (time constant 5(-10 ms); and phase 4, an equal and opposite increase (several seconds). Phase 3 is abolished by curare and does not depend on the history of the membrane voltage. We consider several mechanisms for phases 3 and 4.     

A photoisomerizable muscarinic antagonist. Studies of binding and of conductance relaxations in frog heart

These experiments employ the photoisomerizable compound, 3,3'-bis- [alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans- Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 microM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltage-clamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (less than 1 ms duration) from a xenon flashlamp. Trans leads to cis and cis leads to trans photoisomerizations cause small (less than 20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23 degrees C, k is approximately 3 s-1 and n is 2. Neither k nor n is affected when: (a) [Bis-Q] is varied between 5 and 100 microM; (b) [carbachol] is varied between 1 and 50 microM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-beta-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30 degrees C, k increases with temperature; the Q10 is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy.     

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

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

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Voltage dependence of acetylcholine receptor channel gating in rat myoballs

Whole-cell currents from nicotinic acetylcholine receptor (AChR) channels were studied in rat myoballs using a light-activated agonist to determine the voltage dependence of the macroscopic opening and closing rate constants. Myoballs were bathed in a solution containing a low concentration of the inactive isomer of the photoisomerizable azobenzene derivative, cis-Bis-Q. A light flash was then presented to produce a known concentration jump of agonist, trans-Bis-Q, across a wide range of membrane potentials in symmetrical solutions (NaCl or CsCl on both sides) or asymmetrical solutions (NaCl in the bath and CsCl in the pipette). At the low agonist concentration used in this study, the reciprocal of the macroscopic time constants gives an unambiguous measure of the effective closing rate. It showed an exponential decrease with membrane hyperpolarization between +20 and -100 mV, but tended to level off at more depolarized and at more hyperpolarized membrane potentials. The relative effective opening rate was derived from the steady-state conductance, the single-channel conductance, and the apparent closing rate; it decreased sharply in the depolarizing region and tended to level off and then turn up in the hyperpolarizing region. The two effective rate constants were shown to depend on the first, second, and third power of membrane potential.     

Voltage transients from photo-isomerizing azo dye in bilayer membranes.

Voltage transients are induced by brief light flashed on bilayer membranes with absorbed 3,3'-bis(alpha-(trimethylammonium)methyl)azobenzene (Bis-Q). The voltages are positive for trans-to-cis photo-isomerization, and negative for cis-to-trans photo-isomerization. The risetimes in phosphatidylethanolamine-decane bilayer membranes indicate that absorbed trans-Bis-Q is photo-isomerized to cis within 2 microseconds, and that cis is photo-isomerized to trans within 15 microseconds.     

Evidence for cooperativity between nicotinic acetylcholine receptors in patch clamp records

It is often assumed that ion channels in cell membrane patches gate independently. However, in the present study nicotinic receptor patch clamp data obtained in cell-attached mode from embryonic chick myotubes suggest that the distribution of steady-state probabilities for conductance multiples arising from concurrent channel openings may not be binomial. In patches where up to four active channels were observed, the probabilities of two or more concurrent openings were greater than expected, suggesting positive cooperativity. For the case of two active channels, we extended the analysis by assuming that 1) individual receptors (not necessarily identical) could be modeled by a five-state (three closed and two open) continuous-time Markov process with equal agonist binding affinity at two recognition sites, and 2) cooperativity between channels could occur through instantaneous changes in specific transition rates in one channel following a change in conductance state of the neighboring channel. This allowed calculation of open and closed sojourn time density functions for either channel conditional on the neighboring channel being open or closed. Simulation studies of two channel systems, with channels being either independent or cooperative, nonidentical or identical, supported the discriminatory power of the optimization algorithm. The experimental results suggested that individual acetylcholine receptors were kinetically identical and that the open state of one channel increased the probability of opening of its neighbor.     

Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor.     

The structure and function of glutamate receptor ion channels

As in the case of many ligand-gated ion channels, the biochemical and electrophysiological properties of the ionotropic glutamate receptors have been studied extensively. Nevertheless, we still do not understand the molecular mechanisms that harness the free energy of agonist binding, first to drive channel opening, and then to allow the channel to close (desensitize) even though agonist remains bound. Recent crystallographic analyses of the ligand-binding domains of these receptors have identified conformational changes associated with agonist binding, yielding a working hypothesis of channel function. This opens the way to determining how the domains and subunits are assembled into an oligomeric channel, how the domains are connected, how the channel is formed, and where it is located relative to the ligand-binding domains, all of which govern the processes of channel activation and desensitization.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes

Lidocaine is a local anaesthetic that blocks sodium channels, but also inhibits several ligand-gated ion-channels. The aim of this work was to unravel the mechanisms by which lidocaine blocks Torpedo nicotinic receptors transplanted to Xenopus oocytes. Acetylcholine-elicited currents were reversibly blocked by lidocaine, in a concentration dependent manner. At doses lower than the IC(50) , lidocaine blocked nicotinic receptors only at negative potentials, indicating an open-channel blockade; the binding site within the channel was at about 30% of the way through the electrical field across the membrane. In the presence of higher lidocaine doses, nicotinic receptors were blocked both at positive and negative potentials, acetylcholine dose-response curve shifted to the right and lidocaine pre-application, before its co-application with acetylcholine, enhanced the current inhibition, indicating all together that lidocaine also blocked resting receptors; besides, it increased the current decay rate. When lidocaine, at low doses, was co-applied with 2-(triethylammonio)-N-(2,6-dimethylphenyl) acetamide bromide, edrophonium or 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, which are quaternary-ammonium molecules that also blocked nicotinic receptors, there was an additive inhibitory effect, indicating that these molecules bound to different sites within the channel pore. These results prove that lidocaine blocks nicotinic receptors by several independent mechanisms and evidence the diverse and complex modulation of this receptor by structurally related molecules.     

Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15 degrees C the rate constant, α, equals 1.2 ms(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10(7) to 2x10(8) M(-1)s(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10(2) to 3x10(3)s(-1).     

Arginine 222 in the pre-transmembrane domain 1 of 5-HT3A receptors links agonist binding to channel gating

Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT3A receptors and alpha7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wild-type and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT3 receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT3A receptors.     

Dual modulation of a potassium channel by the m1 muscarinic and beta2-adrenergic receptors

Neurotransmitter receptors alter membrane excitability and synaptic efficacy by generating intracellular signals that ultimately change the properties of ion channels. Given their critical role in controlling cell membrane potential, potassium channels are frequently the targets of modulatory signals from many different G protein-coupled receptors. However, due to the heterogeneity of potassium channel expression in vivo, it has been difficult to determine the molecular mechanisms governing the regulation of molecularly defined potassium channels. Through expression studies in Xenopus oocytes and mammalian cells, we found that the m1 muscarinic acetylcholine receptor (mAChR) potently suppresses a cloned delayed rectifier potassium channel, termed RAK, through a pathway involving phospholipase C activation and direct tyrosine phosphorylation of the RAK protein. In contrast, we found that RAK channel activity is strongly enhanced following agonist activation of beta2-adrenergic receptors; this effect requires a single PKA consensus phosphorylation site located near the amino terminus of the channel protein. These results demonstrate that a specific type of potassium channel that is widely expressed in the mammalian brain and heart is subject to both positive and negative regulation by G protein-dependent pathways.     

Inactivation of monazomycin-induced voltage-dependent conductance in thin lipid membranes. II. Inactivation produced by monazomycin transport through the membrane

At sufficiently large conductances, the voltage-dependent conductance induced in thin lipid membranes by monazomycin undergoes inactivation. This is a consequence of depletion of monazomycin from the membrane solution interface, as monazomycin crosses the membrane to the opposite (trans) side from which it was added. The flux of monazomycin is directly proportional to the monazomycin-induced conductance; at a given conductance it is independent of monazomycin concentration. We conclude that when monazomycin channels break up, some or all of the molecules making up a channel are deposited on the trans side. We present a model for the monazomycin channel: approximately five molecules, each spanning the membrane with its NH3+ on the trans side and an uncharged hydrophilic (probably sugar) group anchored to the cis side, form an aqueous channel lined by--OH groups. The voltage dependence arises from the flipping by the electrical field of molecules lying parallel to the cis surface into the "spanned state;" the subsequent aggregation of these molecules into channels is, to a first approximation, voltage independent. The channel breakup that deposits monomers on the trans side involves the collapsing of the channel in such a way that the uncharged hydrophilic groups remain in contact with the water in the channel as they close the channel from behind. We also discuss the possibility that inactivation of sodium channels in nerve involves the movement from one side of the membrane to the other of the molecules (or molecule) forming the channel.     

Sidedness of reconstituted calcium channels from muscle transverse tubules as determined by D600 and D890 blockade.

The verapamil-type calcium antagonist, D600, and its charged quaternary derivative, D890, were used to assess the sidedness of blockade in single calcium channels reconstituted from purified transverse tubules of skeletal muscle. Spontaneous single channel openings were induced with the agonist Bay-K8644 and recordings were made in a two-chamber planar bilayer setup so that drugs could be delivered to either side of the channel. Micromolar drug addition resulted in a greater than 10-fold decrease in probability of open channel events (po) without a significant change in single channel currents. Changes in po occurred in parallel with changes in mean open time and both parameters could be titrated with a similar IC50. At pH 7.2, cis or trans D600 blocked with an IC50 of 5 microM but for D890 the IC50 was cis 3 microM and trans greater than 75 microM (cis is the intracellular-equivalent side as defined by the voltage-dependent activation). The asymmetry of D890 blockade indicates that the drug can readily gain access to the blocking site from the aqueous phase adjacent to the inner but not extracellular end of the channel.     

Ultrafast conformational dynamics in cyclic azobenzene peptides of increased flexibility

Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the cis to trans and the trans to cis photoreactions. For the cis to trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the trans to cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps-1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10 ns.     

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.