Developmental changes in transmitter sensitivity and synaptic transmission in embryonic chicken sympathetic neurons innervated in vitro.

Dispersed neurons from embryonic chicken sympathetic ganglia were innervated in vitro by explants of spinal cord containing the autonomic preganglionic nucleus or somatic motor nucleus. The maturation of postsynaptic acetylcholine (ACh) sensitivity and synaptic activity was evaluated from ACh and synaptically evoked currents in voltage-clamped neurons at several stages of innervation. All innervated cells are more sensitive to ACh than uninnervated neurons regardless of the source of cholinergic input. Similarly, medium conditioned by either dorsal or ventral explants mimics innervation by enhancing neuronal ACh sensitivity. This increase is due to changes in the rate of appearance of ACh receptors on the cell surface. There are also several changes in the nature of synaptic transmission with development in vitro, including an increased frequency of synaptic events and the appearance of larger amplitude synaptic currents. In addition, the mean amplitude of the unit synaptic current mode increases, as predicted from the observed changes in postsynaptic sensitivity. Although spontaneous synaptic current amplitude histograms with multimodal distributions are seen at all stages of development, histograms from early synapses are typically unimodal. Changes in the synaptic currents and ACh sensitivity between 1 and 4 days of innervation were paralleled by an increase in the number of synaptic events that evoked suprathreshold activity in the postsynaptic neurons. The early pre- and postsynaptic differentiation described here for interneuronal synapses formed in vitro may be responsible for increased efficacy of synaptic transmission during development in vivo.     

Single channel properties of newly synthesized acetylcholine receptors following denervation of mammalian skeletal muscle

We have examined the single channel properties of newly synthesized acetylcholine (ACh) receptors in denervated adult mouse muscle. Patch-clamp recordings were made on freshly isolated fibers from flexor digitorum brevis (fdb) muscles that had been denervated in vivo for periods up to 3 wk. Muscles were treated with alpha-bungarotoxin (alpha-BTX), immediately before denervation, in order to block pre-existing receptors. Denervated fibers exhibited two types of ACh receptor channels, which differed in terms of single channel conductance (45 and 70 pS) and mean channel open time (approximately 7 and 2.5 ms, respectively). In contrast to innervated muscle, where only 3% of the total openings were contributed by the low-conductance channel type, greater than 80% of the openings in the nonsynaptic membrane of denervated muscle were of this type. Importantly, a similar increase in the proportion of low-conductance channels was observed for recordings from synaptic membrane after denervation. These data argue against the proposal that, in denervated muscle, the low-conductance channels undergo continued conversion to the high-conductance type focally at the site of former synaptic contact. Rather, our findings provide additional support for the idea that the functional properties of ACh receptors are governed uniformly by the state of innervation of the fiber and not by proximity to the site of synaptic contact.     

Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G-proteins to activate ion channels.

Following the infection of insect ovarian cells (Sf9) with recombinant bearing the cDNA coding for the rat muscarinic acetylcholine (ACh) receptor subtype m3, ionic flux across the membrane in response to the application of ACh was examined electrophysiologically. We show that ACh activates potassium currents. The response is abolished when cells are treated with pertussis toxin. No ACh-induced currents are observed from uninfected cells or cells infected with virus which do not contain the cDNA coding for ACh receptors in its genome. The characteristics of single channel currents show time-dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 pS. The conductance level of channels gradually increases in steps to 10 pS and then to 20 pS and 40 pS. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synchrony with, the original channel.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.     

Some properties of acetylcholine receptors in human cultured myotubes

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [125I]alpha-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 +/- 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40-45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 microM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. -70 mV, 22 degrees C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 microM). With desensitizing doses of ACh (10 microM), channel openings occurred in obvious bursts; each burst usually appeared as part of a 'cluster' of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.     

Functional properties of acetylcholine receptors coexpressed with the 43K protein in heterologous cell systems.

The nicotinic acetylcholine (ACh) receptor is an integral membrane protein which mediates synaptic transmission at the skeletal neuromuscular junction. A key event in the development of the neuromuscular junction is the formation of high density aggregates of ACh receptors in the postsynaptic membrane. Receptor clustering has been attributed, in part, to their association with a peripheral membrane protein of Mr 43,000 (43K protein). We have addressed whether the association of the 43K protein can alter the single channel properties of the ACh receptor, and thus influence neuromuscular transmission at developing synapses, by expressing ACh receptors with and without the 43K protein in heterologous expression systems. We found that coexpression of the 43K protein with the receptor did not significantly alter either its single channel conductance or its mean channel open time. This was true in oocytes and also in COS cells where it was possible to localize 43K-induced clusters by fluorescence microscopy and to record from those clustered receptors. These data are in agreement with previous single channel studies which have shown that the properties of diffusely distributed and clustered receptors in native muscle cells from both mice and Xenopus do not differ.     

Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and ?80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.     

A physiological study on acetylcholine receptor expressed in Xenopus oocytes from cloned cDNAs

Nicotinic ACh receptor was expressed in Xenopus oocytes by injecting mRNAs produced from cloned cDNAs encoding the four subunits of ACh receptor of Torpedo californica. ACh responses recorded from oocytes 3 days after injection of the mRNAs were reversibly blocked by d-tubocurarine (1-2 microM), indicating that the newly synthesized receptor is of nicotinic type. The reversal potential of ACh response was found at around -1 - -5 mV. The reversal potential was not changed by removal of extracellular C1-, suggesting that the ionic channel of the newly expressed ACh receptor is permeable only to cations. Repetitive applications of ACh caused desensitization of the receptor. The rate of the desensitization was greater when the membrane potential was more negative. Subunit deletion studies showed that all four subunits are required for the formation of ACh receptors with normal ACh sensitivity. However, ACh receptors without delta subunit responded to ACh with low sensitivity. Studies on ACh receptor mutants with -subunits altered by site directed mutagenesis of the cDNA suggest that the anphipathic segment is involved in the channel function of the receptor as well as the four hydrophobic segments since partial deletion of amino acids in these segments essentially abolished ACh sensitivity with relatively little change in 125I-alpha-bungarotoxin binding activity.     

Regulation of acetylcholine receptor channel function during development of skeletal muscle

The nicotinic acetylcholine (ACh) receptor channel mediates synaptic transmission at the neuromuscular junction. During the development of skeletal muscle, ACh receptors undergo changes in distribution, antigenic determinants, degradation rate, and function. Now that these developmental hallmarks have been identified, attention has turned toward understanding both the structural bases for such changes and the role of nerve in triggering these changes. Recently, a much clearer understanding of one of these developmental processes, namely, the alterations in channel function, has emerged through both sensitive patch-clamp measurements and the application of recombinant DNA technology. In light of these new advances, we now reevaluate the processes governing the developmental changes in the functional properties of the ACh receptor.     

Development of physiological responses to acetylcholine during organ culture of young embryonic chick hearts

Young (3 days-old) embryonic chick hearts (i.e., prior to innervation) have a low sensitivity and transient response to acetylcholine (ACh). In the present experiments, in which the hearts were placed into organ culture for a period of up to 7 days, the sensitivity to ACh was found to increase with time in culture, and the inhibitory action of ACh persisted for a longer time (i.e., desensitization declined). Thus, the ability to ACh to slow spontaneous heart rate and to shorten action potential duration (in the atrium) became greater during the course of organ culture. The effects of ACh on spontaneous heart rate and on action potential duration were blocked by atropine. These results suggest that the physiological responses to muscarinic agonists in young embryonic chick hearts continue to develop in organ culture, and that the increased sensitivity to muscarinic agonists seen in ovo is unlikely to be due to the development of innervation.     

The M2 muscarinic G-protein-coupled receptor is voltage-sensitive

G-protein coupled receptors are not considered to exhibit voltage sensitivity. Here, using Xenopus oocytes, we show that the M2 muscarinic receptor (m2R) is voltage-sensitive. The m2R-mediated potassium channel (GIRK) currents were used to assay the activity of m2R. We found that the apparent affinity of m2R toward acetylcholine (ACh) was reduced upon depolarization. Binding experiments of [3H]ACh to individual oocytes expressing m2R confirmed the electrophysiological findings. When the GIRK channels were activated either by overexpression of Gbetagamma subunits or by injection of GTPgammaS, the ratio between the currents measured at -60 mV and +40 mV was the same as for the basal activity of the GIRK channel. Thus, the steps downstream to agonist activation of m2R are not voltage-sensitive. We further found that, in contrast to m2R, the apparent affinity of m1R was increased upon depolarization. We also found that the voltage sensitivity of binding of [3H]ACh to oocytes expressing m2R was greatly diminished following pretreatment with pertussis toxin. The cumulative results suggest that m2R is, by itself, voltage-sensitive. Furthermore, the voltage sensitivity does not reside in the ACh binding site, rather, it most likely resides in the receptor region that couples to the G-protein.     

Molecular basis of the differential sensitivity of nematode and mammalian muscle to the anthelmintic agent levamisole

Levamisole is an anthelmintic agent that exerts its therapeutic effect by acting as a full agonist of the nicotinic receptor (AChR) of nematode muscle. Its action at the mammalian muscle AChR has not been elucidated to date despite its wide use as an anthelmintic in humans and cattle. By single channel and macroscopic current recordings, we investigated the interaction of levamisole with the mammalian muscle AChR. Levamisole activates mammalian AChRs. However, single channel openings are briefer than those activated by acetylcholine (ACh) and do not appear in clusters at high concentrations. The peak current induced by levamisole is about 3% that activated by ACh. Thus, the anthelmintic acts as a weak agonist of the mammalian AChR. Levamisole also produces open channel blockade of the AChR. The apparent affinity for block (190 microm at -70 mV) is similar to that of the nematode AChR, suggesting that differences in channel activation kinetics govern the different sensitivity of nematode and mammalian muscle to anthelmintics. To identify the structural basis of this different sensitivity, we performed mutagenesis targeting residues in the alpha subunit that differ between vertebrates and nematodes. The replacement of the conserved alphaGly-153 with the homologous glutamic acid of nematode AChR significantly increases the efficacy of levamisole to activate channels. Channel activity takes place in clusters having two different kinetic modes. The kinetics of the high open probability mode are almost identical when the agonist is ACh or levamisole. It is concluded that alphaGly-153 is involved in the low efficacy of levamisole to activate mammalian muscle AChRs.     

Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.     

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).     

Role of voltage-sensitive receptors in nicotinic transmission.

This paper compares the conductance induced by bath-applied acetyl-choline (ACh) and by the same transmitter released from nerve terminals at Electrophorus electroplaques. For the former case, dose-response relations are characterized by the maximal agonist-induced conductance, rgamma (130 mmho/cm2), and by the concentration which induces half this conductance; this concentration is termed Kapp and equals 50 micron at -85 mV. For the latter case, neurally evoked postsynaptic currents (PSCs) are characterized by the peak conductance during strongly facilitated release, gPSC, and by the rate constant for decay, alpha. Since gPSC roughly equals rgamma, it is concluded that the PSC activates nearly all available receptor channels. These and other data agree with recent estimates that during the growth phase of the quantal response, (a) the ACh concentration is at least several hundred micromolar; and (b) most nearby channels are activated. However both alpha and Kapp increase during depolarization, at a rate of about e-fold per 86 mV. These observations on voltage sensitivity suggest that a suprathreshold synaptic event is rapidly terminated because the action potential abruptly releases ACh molecules from receptors.     

Effect of curare on responses to different putative neurotransmitters in Aplysia neurons.

We have studied the effects of curare on responses resulting from iontophoretic application of several putative neurotransmitters onto Aplysia neurons. These neurons have specific receptors for acetylcholine (ACh), dopamine, octopamine, phenylethanolamine, histamine, gamma-aminobutyric acid (GABA), aspartic acid, and glutamic acid. Each of these substances may on different specific neurons elicit at least three types of response, caused by a fast depolarizing Na+, a fast hyperpolarizing Cl-, or a slow hyperpolarizing K+ conductance increase. All responses resulting from either Na+ or Cl- conductance increases, irrespective of which putative transmitter activated the response, were sensitive to curare. Most were totally blocked by less than or equal to 10-4 M curare. GABA responses were less sensitive and were often only depressed by 10-3 M curare. K+ conductance responses, irrespective of the transmitter, were not curare sensitive. These results are consistent with a model of receptor organization in which one neurotransmitter receptor may be associated with any of at least three ionophores, mediating conductance increase responses to Na+, Cl-, and K+, respectively. In Aplysia nervous tissue, curare appears not to be a specific antagonist for the nicotinic ACh receptor, but rather to be a specific blocking agent for a class of receptor-activated Na+ and Cl- responses.     

Calcium dependence of acetylcholine induced conductance changes.

The influence of external calcium variation (0.7–28 mM) on acetylcholine (ACh) induced conductance changes was examined under voltage clamp conditions in snail neurons in which ACh elicitis hyperpolarizing responses and in which the main current-carrying ion species is Cl^?. Raising external calcium to 28 mM as well as lowering external calcium to 1.75 mM decreased the ACh-induced condutance change without altering the reversal potential for ACh-induced currents. Lowering external calcium to 0.7 mM increased the ACh-induced conductance change and shifted the reversal potential to less negative values. The results at 28 mM calcium can be best explained on the assumption that excess calcium interferes with the interaction between ACh and the receptor. The results at 1.75 mM calcium can be satisfactorily explained in terms of the concept that fixed membrane charges play a role in regulating Cl^? permeation through transmitter sensitive membranes. Evidence was also obtained that in snail neurons receptor inactivation increases when external calcium is reduced.     

Nicotinic acetylcholine receptors assembled from the alpha7 and beta3 subunits.

Intracellular recordings were performed in voltage-clamped Xenopus oocytes upon injection with a mixture of cDNAs encoding the beta3 and mutant alpha7 (L247Talpha7) neuronal nicotinic acetylcholine receptor (nAChR) subunits. The expressed receptors maintained sensitivity to methyllycaconitine and to alpha-bungarotoxin but exhibited a functional profile strikingly different from that of the homomeric L247Talpha7 receptor. The heteromeric L247Talpha7beta3 nAChR had a lower apparent affinity and a faster rate of desensitization than L247Talpha7 nAChR, exhibited nonlinearity in the I-V relationship, and was inhibited by 5-hydroxytryptamine, much like wild type alpha7 (WTalpha7) nAChR. Single channel recordings in cell-attached mode revealed unitary events with a slope conductance of 19 picosiemens and a lifetime of 5 ms, both values being much smaller than those of the homomeric receptor channel. Upon injection with a mixture of WTalpha7 and beta3 cDNAs, clear evidence was obtained for the plasma membrane assembly of heteromeric nAChRs, although ACh could not activate these receptors. It is concluded that beta3, long believed to be an orphan subunit, readily co-assembles with other subunits to form heteromeric receptors, some of which may be negative regulators of cholinergic function.