Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.     

Quantal analysis indicates an alpha-toxin-like block by lophotoxin, a non-ionic marine natural product

The effects of a structurally novel paralytic substance (lophotoxin) on quantal transmission parameters and the time course of synaptic potentials have been examined. This substance completely abolished potentials by reducing quantal size without affecting the release of quanta. Nerve conduction, membrane potential, and the passive electrical properties of the muscle end-plate remained unaffected. Lophotoxin appears to act directly on the acetylcholine receptor-channel complex, although perhaps not the cholinoreceptive site itself, as suggested by the unusual chemistry and onset kinetics of this toxin.     

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

Single Channel Kinetics of a Glutamate Receptor

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the presence of 10^-4 M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

On the mechanism of a mammalian neuronal type nicotinic acetylcholine receptor investigated by a rapid chemical kinetic technique. Detection and characterization of a short-lived, previously unobserved, main receptor form in PC12 cells.

The mammalian nicotinic acetylcholine receptor in PC12 cells has many properties characteristic of the neuronal receptors involved in key chemical reactions that are responsible for signal transmission between cells of the nervous system. This report describes initial investigations of the mechanism of this receptor using a rapid chemical kinetic technique with a time resolution of 20 ms, which represents a 250-fold improvement over the best time resolution (5 s) employed in previous studies. Carbamoylcholine, a stable analogue of the neurotransmitter acetylcholine, was the activating ligand used, and the concentration of open transmembrane receptor-channels in PC12 cells was measured by recording whole-cell currents at pH 7.4, 21-23 degrees C, and a transmembrane voltage of -60 mV. Two receptor forms that account for 80% and 20% of the receptor-controlled current were detected; the main receptor form, accounting for 80% of the whole-cell current, desensitized completely before the first measurements had been made in previous studies. Only the main receptor form has been investigated so far using the new method. The constants of a mechanism that accounts for the concentration of the open transmembrane receptor-channel over a 100-fold range of carbamoylcholine concentration were evaluated: the dissociation constant of the site controlling channel opening (K1 = 2.0 mM), the channel-opening equilibrium constant (phi -1 = 5.0), and the dissociation constant of an inhibitory site to which carbamoylcholine binds (KR = 6.5 mM). These evaluated constants allow one to calculate Po, the conditional probability that at a given concentration of carbamoylcholine the receptor-channel is open. Po was also determined in the presence of 2 mM carbamoylcholine by an independent method, the single-channel current-recording technique, and the agreement between the Po values obtained in two independent ways is within experimental error. This result indicates that the time resolution of the chemical kinetic technique employed was sufficient to evaluate the constants pertaining to the active state of the receptor, which forms a transmembrane channel, before its conversion to desensitized receptor forms with different properties. Previous kinetic measurements with a time resolution of 5 s showed that many compounds, such as anesthetic-like molecules, nerve growth factor, and substance P, modify the function of the neuronal receptor in PC12 cells or react specifically with the neuronal but not with the muscle receptor, for example, some toxins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of unliganded acetylcholine receptor channel gating.

Open- and closed-state lifetimes of unliganded acetylcholine receptor channel activity were analyzed by the method of likelihood maximazation. For both open times and closed times, the best-fitting density is most often a sum of two exponentials. These multiple open states cannot depend on the number of receptor binding sites occupied since they are observed in the absence of ligand. The rate of spontaneous opening and the faster decay constant of closing increased as the membrane was hyperpolarized. The voltage dependence of the rate of spontaneous opening is stronger than that for curare-liganded channels. Evidence that the acetylcholine receptor channel can open spontaneously in the absence of ligand has been presented previously (Sanchez et al, 1983; Brehm et al, 1984; Jackson, 1984). To add to this evidence, alpha-bungarotoxin was added to the patch electrode, causing the frequency of openings to decay with time. The rate constant determined from this decay is similar to rate constants reported for the binding of iodinated alpha-bungarotoxin to the acetylcholine receptor. The frequency of unliganded channel opening has been estimated as 2 X 10(-3) s-1 per receptor. A comparison of carbamylcholine-liganded and spontaneous gating transition rates suggests that ligand binding increases the rate of opening by a factor of 1.4 X 10(7). Carbamylcholine binding increases the mean open time by a factor of 5. Thus, a cholinergic agonist activates the acetylcholine receptor by destabilizing the closed state. The liganded and unliganded channel gating rates were used to analyze the energetics of ligand activation of the acetylcholine receptor channel, and to relate the open channel dissociation constant to the closed channel dissociation constant.     

Characterization of the channel properties of a neuronal acetylcholine receptor reconstituted into planar lipid bilayers

An alpha-toxin-binding membrane protein, isolated from the head and thoracic ganglia of the locus (Locusta migratoria), was reconstituted into planar lipid bilayers. Cholinergic agonists such as acetylcholine, carbamylcholine, and suberyldicholine induced fluctuations of single channels, which suggests that the protein represents a functional cholinergic receptor channel. The antagonist d-tubocurarine blocked the activation of the channels, whereas hexamethonium had only a weak effect; similar properties have been described for nicotinic insect receptors in situ. The channel was selectively permeable to monovalent cations but was impermeable to anions. The conductance of the channel (75 pS in 100 mM NaCl) was independent of the type of agonist used to activate the receptor. Kinetic analysis of the channel gating revealed that, at high agonist concentrations (50 microM carbamylcholine), more than one closed state exists and that multiple gating events, bursting as well as fast flickering, appeared. At very high agonist concentrations (500 microM carbamylcholine), desensitization was observed. Channel kinetics were dependent on the transmembrane potential. Comparing the conductance, the kinetics, and the pharmacology of nicotinic acetylcholine receptor from insect ganglia and fish electroplax reconstituted into bilayers revealed obvious similarities but also significant differences.     

Modulation of proton-induced current fluctuations in the human nicotinic acetylcholine receptor channel

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that switches upon activation from a closed state to a full conducting state. We found that the mutation delta S268K, located at 12' position of the second transmembrane domain of the delta subunit of the human nAChR generates a long-lived intermediate conducting state, from which openings to a wild-type like conductance level occur on a submillisecond time scale. Aiming to understand the interplay between structural changes near the 12' position and channel gating, we investigated the influence of various parameters: different ligands (acetylcholine, choline and epibatidine), ligand concentrations, transmembrane voltages and both fetal and adult nAChRs. Since sojourns in the high conductance state are not fully resolved in time, spectral noise analysis was used as a complement to dwell time analysis to determine the gating rate constants. Open channel current fluctuations are described by a two-state Markov model. The characteristic time of the process is markedly influenced by the ligand and the receptor type, whereas the frequency of openings to the high conductance state increases with membrane hyperpolarization. Conductance changes are discussed with regard to reversible transfer reaction of single protons at the lysine 12' side chain.     

Parallel Postnatal Development of Choline Acetyltransferase Activity and Muscarinic Acetylcholine Receptors in the Rat Olfactory Bulb

The development of cholinergic synapses in the rat olfactory bulb was investigated by measuring changes in the activity of choline acetyltransferase (ChAT; EC 2.3.1.6.), a presynaptic cholinergic marker, and in the concentration of muscarinic receptors, components of cholinoceptive membranes. Three biochemical properties of the muscarinic system also were examined for possible differentiation: ligand binding, molecular weight, and isoelectric point. Receptors from embryonic (day 18), neonatal (postnatal day 3), and adult rat olfactory bulbs exhibited identical complex binding (nH = 0.45) of the agonist carbachol. For each age, the relative proportions of high-affinity (Ki approximately equal to 1.0 microM) and low-affinity (Ki approximately equal to 100 microM) binding states were 60% and 40%, respectively. The antagonist pirenzepine also bound to high-affinity (Ki approximately equal to 0.15 microM, RH approximately equal to 70%) and low-affinity (Ki approximately equal to 2.0 microM, RL approximately equal to 30%) sites in neonatal and adult rats. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis of [3H]propylbenzilylcholine mustard-labeled receptors from neonatal and adult rats showed a single electrophoretic form with an apparent molecular weight of 65,000. In contrast, analytical isoelectric focusing indicated high pI (4.50) and low pI (4.00) receptor forms were present. Neonatal rats contained approximately equal proportions of the two receptor forms, whereas adult rats contained mainly the low pI form, indicating that molecular alteration of the receptor population had occurred during development. Comparison of postnatal changes in acetylcholine receptors and ChAT activity showed a striking correlation between the development of cholinergic terminals and muscarinic receptors. Throughout the first postnatal week, ChAT activity remained at 5% of adult levels; activity began to rise on postnatal day 6 and gradually reached adult levels (56 +/- 4 mumol of [3H]acetylcholine/h/g) during the fourth week. Similarly, muscarinic receptor concentration was low (30-50 fmol/mg) throughout the first week, began to rise at postnatal day 7; and reached 90% of adult levels (317 +/- 17 fmol/mg) by the fourth week. In contrast, there was little increase in the concentration of nicotinic acetylcholine receptors (30 fmol/mg) during this period. The parallel postnatal development of ChAT activity and muscarinic receptors suggests the existence of factors that couple the differentiation of presynaptic cholinergic terminals and postsynaptic cholinoceptive elements.     

A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.     

Modulation of proton-induced current fluctuations in the human nicotinic acetylcholine receptor channel

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that switches upon activation from a closed state to a full conducting state. We found that the mutation δ S268K, located at 12′ position of the second transmembrane domain of the δ subunit of the human nAChR generates a long-lived intermediate conducting state, from which openings to a wild-type like conductance level occur on a submillisecond time scale. Aiming to understand the interplay between structural changes near the 12′ position and channel gating, we investigated the influence of various parameters: different ligands (acetylcholine, choline and epibatidine), ligand concentrations, transmembrane voltages and both fetal and adult nAChRs. Since sojourns in the high conductance state are not fully resolved in time, spectral noise analysis was used as a complement to dwell time analysis to determine the gating rate constants. Open channel current fluctuations are described by a two-state Markov model. The characteristic time of the process is markedly influenced by the ligand and the receptor type, whereas the frequency of openings to the high conductance state increases with membrane hyperpolarization. Conductance changes are discussed with regard to reversible transfer reaction of single protons at the lysine 12′ side chain.     

The use of dwell time cross-correlation functions to study single-ion channel gating kinetics.

The derivation of cross-correlation functions from single-channel dwell (open and closed) times is described. Simulation of single-channel data for simple gating models, alongside theoretical treatment, is used to demonstrate the relationship of cross-correlation functions to underlying gating mechanisms. It is shown that time irreversibility of gating kinetics may be revealed in cross-correlation functions. Application of cross-correlation function analysis to data derived from the locust muscle glutamate receptor-channel provides evidence for multiple gateway states and time reversibility of gating. A model for the gating of this channel is used to show the effect of omission of brief channel events on cross-correlation functions.     

Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle

We used specific antibodies to gamma, delta, and epsilon subunits to characterize acetylcholine receptor (AChR) in extracts and at endplates of developing, adult, and denervated rat muscle. The AChRs in normal adult muscle were immunoprecipitated by anti-epsilon and anti-delta, but not by anti-gamma antibodies, whereas AChRs in denervated and embryonic muscles were precipitated by anti-gamma and anti-delta, but showed little or no reactivity to anti-epsilon antibodies. In immunofluorescence experiments, AChRs at neonatal endplates bound antibodies to gamma or delta, but not epsilon, subunit, whereas those in adult muscles bound antibodies to epsilon or delta, but not gamma, subunit. AChRs at denervated endplates and at developing endplates between postnatal days 9 and 16 bound all three antibodies. We conclude that the distribution of gamma and epsilon subunits of the AChR parallels the distribution of AChRs with embryonic and adult channel properties, respectively.     

Inorganic, monovalent cations compete with agonists for the transmitter binding site of nicotinic acetylcholine receptors.

The properties of adult mouse recombinant nicotinic acetylcholine receptors activated by acetylcholine (ACh+) or tetramethylammonium (TMA+) were examined at the single-channel level. The midpoint of the dose-response curve depended on the type of monovalent cation present in the extracellular solution. The shifts in the midpoint were apparent with both inward and outward currents, suggesting that the salient interaction is with the extracellular domain of the receptor. Kinetic modeling was used to estimate the rate constants for agonist binding and channel gating in both wild-type and mutant receptors exposed to Na+, K+, or Cs+. The results indicate that in adult receptors, the two binding sites have the same equilibrium dissociation constant for agonists. The agonist association rate constant was influenced by the ionic composition of the extracellular solution whereas the rate constants for agonist dissociation, channel opening, and channel closing were not. In low-ionic-strength solutions the apparent association rate constant increased in a manner that suggests that inorganic cations are competitive inhibitors of ACh+ binding. There was no evidence of an electrostatic potential at the transmitter binding site. The equilibrium dissociation constants for inorganic ions (Na+, 151 mM; K+, 92 mM; Cs+, 38 mM) and agonists (TMA+, 0.5 mM) indicate that the transmitter binding site is hydrophobic. Under physiological conditions, about half of the binding sites in resting receptors are occupied by Na+.     

Single-channel autocorrelation functions: the effects of time interval omission.

We present a general mathematical framework for analyzing the dynamic aspects of single channel kinetics incorporating time interval omission. An algorithm for computing model autocorrelation functions, incorporating time interval omission, is described. We show, under quite general conditions, that the form of these autocorrelations is identical to that which would be obtained if time interval omission was absent. We also show, again under quite general conditions, that zero correlations are necessarily a consequence of the underlying gating mechanism and not an artefact of time interval omission. The theory is illustrated by a numerical study of an allosteric model for the gating mechanism of the locust muscle glutamate receptor-channel.     

Open channel block and alteration of N-methyl-D-aspartic acid receptor gating by an analog of phencyclidine.

We investigated inhibition of the N-methyl-D-aspartic acid (NMDA) receptor-channel complex by N-ethyl-1,4,9, 9alpha-tetrahydro-4alphaR-cis-4alphaH-fluoren-++ +4alpha-amine (NEFA), a structural analog of phencyclidine (PCP). Using the whole-cell recording technique, we demonstrated that NEFA inhibits NMDA responses with an IC50 of 0.51 microM at -66 mV. We determined that NEFA binds to the open channel, and subsequently the channel can close and trap the blocker. Once the channel has closed, NEFA is unable to dissociate until the channel reopens. Single-channel recordings revealed that NEFA reduces the mean open time of single NMDA-activated channels in a concentration-dependent manner with a forward blocking rate (k+) of 39.9 microM-1 s-1. A computational model of antagonism by NEFA was developed and constrained using kinetic measurements of single-channel data. By multiple criteria, only models in which blocker binding in the channel causes a change in receptor operation adequately fit or predicted whole-cell data. By comparing model predictions and experimental measurements of NEFA action at a high NMDA concentration, we determined that NEFA affects receptor operation through an influence on channel gating. We conclude that inhibition of NMDA receptors by PCP-like blockers involves a modification of channel gating as well as block of current flow through the open channel.     

Role of pairwise interactions between M1 and M2 domains of the nicotinic receptor in channel gating

The adult form of the nicotinic acetylcholine receptor (AChR) consists of five subunits (alpha(2)betaepsilondelta), each having four transmembrane domains (M1-M4). The atomic model of the nicotinic acetylcholine receptor shows that the pore-lining M2 domains make no extensive contacts with the rest of the transmembrane domains. However, there are several sites where close appositions between segments occur. It has been suggested that the pair alphaM1-F15' and alphaM2-L11' is one of the potential interactions between segments. To determine experimentally if these residues are interacting and to explore if this interhelical interaction is essential for channel gating, we combined mutagenesis with single-channel kinetic analysis. Mutations in alphaM1-F15' lead to profound changes in the opening rate and slighter changes in the closing rate. Channel gating is impaired as the volume of the residue increases. Rate-equilibrium linear free-energy relationship analysis reveals an approximately 70% open-state-like environment for alphaM1-F15' at the transition state of the gating reaction, suggesting that it moves early during the gating process. Replacing the residue at alphaM1-15' by that at alphaM2-11' and vice versa profoundly alters gating, but the combination of the two mutations restores gating to near normal, indicating that alphaM1-F15' and alphaM2-L11' are interchangeable. Double-mutant cycle analysis shows that these residues are energetically coupled. Thus, the interaction between M1 and M2 plays a key role in channel gating.