Amphibian Neuromuscular Junction Research Articles

Seasonal comparison of the neuromuscular junction morphology of Bufo marinus.

At mammalian neuromuscular junctions (NMJs), prolonged inactivity leads to muscle denervation and atrophy. By contrast, amphibian NMJs do not show such degeneration even though they can remain in a state of drought-imposed dormancy (hibernation) for many years. We have previously reported that during the dry season, toad (Bufo marinus) NMJs display decreased sensitivity to extracellular calcium-dependent neurotransmitter release, which leads to minimal neuromuscular transmission. In the present study, we examined and compared NMJ morphology of toads obtained from the wild during the wet season (February-March) when these toads are active, to toads obtained from dry season (October-November) when toads are inactive. Iliofibularis muscles were isolated and prepared for immunostaining with anti-SV2, a monoclonal antibody that labels synaptic vesicle glycoprotein SV2. The corresponding postsynaptic acetylcholine receptors were stained using Alexa Fluro-555 conjugated α-bungarotoxin. Confocal microscopy and three-dimensional reconstructions were then used to examine the pre-and postsynaptic morphology of toads NMJs from the dry (inactive) and wet (active) seasons. Total axon branch number, the percentage of axon branches with discontinuous distributions of synaptic vesicles, and further the Pearson value of colocalization of pre and postsynaptic elements in each NMJs from both the dry and wet season were compared. While our previous studies on dry toads revealed a significant reduction in evoked neurotransmission, our present findings show that the structure of the NMJs suffered limited level of remodeling, suggesting a mechanism utilized by NMJs in dry season toads to support quick recover from their dormant state after the heavy rain in wet season.

Seasonal factors influence quantal transmitter release and calcium dependence at amphibian neuromuscular junctions.

Amphibian neuromuscular junctions (NMJs) are composed of hundreds of neurotransmitter release sites that exhibit nonuniform transmitter release probabilities and demonstrated seasonal modulation. We examined whether recruitment of release sites is variable when the extracellular calcium concentration ([Ca2+]o) is increased in the wet and dry seasons. The amount of transmitter released from the entire nerve terminal increases by approximately the fourth power as [Ca2+]o is increased. Toad (Bufo marinus) NMJs were visualized using 3,3'-diethyloxardicarbocyanine iodide [DiOC2(5)] fluorescence, and focal loose patch extracellular recordings were used to record the end-plate currents (EPCs) from small groups of release sites. Quantal content (m̄e ), average probability of quantal release (pe ), and the number of active release sites (ne ) were determined for different [Ca2+]o Our results indicated that the recruitment of quantal release sites with increasing [Ca2+]o differs spatially (between different groups of release sites) and also temporally (in different seasons). These differences were reflected by the nonuniform alterations in pe and ne Most release site groups demonstrated an increase in both pe and ne when [Ca2+]o increased. In ~30% of release site groups examined, pe decreased while ne increased only during the active period (wet season). Although the dry season induced parallel right shift in the quantal release versus extracellular calcium concentration when compared with the wet season, the dependence of quantal content on [Ca2+]o was not changed. These results demonstrate the flexibility, reserve, and adaptive capacity of neuromuscular junctions in maintaining appropriate levels of neurotransmission.

Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles.

Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.

Actin- and Dynamin-Dependent Maturation of Bulk Endocytosis Restores Neurotransmission following Synaptic Depletion

Bulk endocytosis contributes to the maintenance of neurotransmission at the amphibian neuromuscular junction by regenerating synaptic vesicles. How nerve terminals internalize adequate portions of the presynaptic membrane when bulk endocytosis is initiated before the end of a sustained stimulation is unknown. A maturation process, occurring at the end of the stimulation, is hypothesised to precisely restore the pools of synaptic vesicles. Using confocal time-lapse microscopy of FM1-43-labeled nerve terminals at the amphibian neuromuscular junction, we confirm that bulk endocytosis is initiated during a sustained tetanic stimulation and reveal that shortly after the end of the stimulation, nerve terminals undergo a maturation process. This includes a transient bulging of the plasma membrane, followed by the development of large intraterminal FM1-43-positive donut-like structures comprising large bulk membrane cisternae surrounded by recycling vesicles. The degree of bulging increased with stimulation frequency and the plasmalemma surface retrieved following the transient bulging correlated with the surface membrane internalized in bulk cisternae and recycling vesicles. Dyngo-4a, a potent dynamin inhibitor, did not block the initiation, but prevented the maturation of bulk endocytosis. In contrast, cytochalasin D, an inhibitor of actin polymerization, hindered both the initiation and maturation processes. Both inhibitors hampered the functional recovery of neurotransmission after synaptic depletion. Our data confirm that initiation of bulk endocytosis occurs during stimulation and demonstrates that a delayed maturation process controlled by actin and dynamin underpins the coupling between exocytosis and bulk endocytosis.

In vivolong-term synaptic plasticity of glial cells

Evidence showing the ability of glial cells to detect, respond to and modulate synaptic transmission and plasticity has contributed to the notion of glial cells as active synaptic partners. However, synaptically induced plasticity of glia themselves remains ill defined. Here we used the amphibian neuromuscular junction (NMJ) to study plasticity of perisynaptic Schwann cells (PSCs), glial cells at this synapse, following long-term in vivo modifications of synaptic activity. We used two models that altered synaptic activity in different manners. First, chronic blockade of postsynaptic nicotinic receptors using alpha-bungarotoxin (alpha-BTx) decreased facilitation, increased synaptic depression and decreased post-tetanic potentiation (PTP). Second, chronic nerve stimulation increased facilitation and resistance to synaptic depression, while leaving PTP unaltered. Our results indicate that there is no direct relationship between transmitter release and PSC calcium responses. Indeed, despite changes in transmitter release and plasticity in stimulated NMJs, nerve-evoked PSC calcium responses were similar to control. Similarly, PSC calcium responses in alpha-BTx treated NMJs were delayed and smaller in amplitude, even though basal level of transmitter release was increased. Also, when isolating purinergic and muscarinic components of PSC calcium responses, we found an increased sensitivity to ATP and a decreased sensitivity to muscarine in chronically stimulated NMJs. Conversely, in alpha-BTx treated NMJs, PSC sensitivity remained unaffected, but ATP- and muscarine-induced calcium responses were prolonged. Thus, our results reveal complex modifications of PSC properties, with differential modulation of signalling pathways that might underlie receptor regulation or changes in Ca(2+) handling. Importantly, similar to neurons, perisynaptic glial cells undergo plastic changes induced by altered synaptic activity.

Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin

Glycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Ca(v)2.2 channels. We found that GLTx stimulates the very high frequency, long-lasting (more than 10 hours) spontaneous release of acetylcholine by promoting nerve terminal Ca(2+) oscillations sensitive to the inhibitor omega-conotoxin GVIA at the amphibian neuromuscular junction. Although an estimate of the number of synaptic vesicles undergoing exocytosis largely exceeds the number of vesicles present in the motor nerve terminal, ultrastructural examination of GLTx-treated synapses revealed no significant change in the number of synaptic vesicles. However, we did detect the appearance of large pre-synaptic cisternae suggestive of bulk endocytosis. Using a combination of styryl dyes, photoconversion and horseradish peroxidase (HRP)-labeling electron microscopy, we demonstrate that GLTx upregulates presynaptic-vesicle recycling, which is likely to emanate from the limiting membrane of these large cisternae. Similar synaptic-vesicle recycling through bulk endocytosis also occurs from nerve terminals stimulated by high potassium. Our results suggest that this process might therefore contribute significantly to synaptic recycling under sustained levels of synaptic stimulation.

Mechanisms of Neuromodulation as Dissected Using Sr2+ at Motor Nerve Endings

The use of binomial analysis as a tool for determining the sites of action of neuromodulators may be complicated by the nonuniformity of release probability. One of the potential sources for nonuniformity of release probability is the presence of multiple forms of synaptotagmins, the Ca2+ sensors responsible for triggering vesicular exocytosis. In this study we have used Sr2+, an ion whose actions may be restricted to a subpopulation of synaptotagmins, in an attempt to obtain meaningful estimates of the binomial parameters p (the probability of evoked acetylcholine [Ach] release) and n (the immediate available store of ACh quanta, whereby m = np). In contrast to results in Ca2+ solutions, binomial analysis of Sr2+-dependent release reveals a dramatically reduced dependence of n on extracellular Sr2+ concentrations. In Sr2+ solutions, blockade of potassium channels with 3,4-diaminopyridine increased m by an exclusive increase in p, whereas treatment with phorbol ester increased m solely by effects on n. The cyclic adenosine monophosphate (cAMP) analogue CPT-cAMP increased m by increasing both n and p. The effect of CPT-cAMP on p but not on n was blocked by protein kinase A (PKA) inhibitors, whereas the effect on n was mimicked by 8-CPT-2'-O-Me-cAMP, a selective agonist for exchange protein directly activated by cAMP, otherwise known as the cAMP-sensitive guanine nucleotide-exchange protein. The results demonstrate both the utility of the binomial distribution in Sr2+ solutions and the dual effects of cyclic AMP on both PKA-dependent and PKA-independent processes at the amphibian neuromuscular junction.

Role for the skeletal muscle action potential in non‐Hebbian long‐term depression at the amphibian (Bufo marinus) neuromuscular junction

Retrograde signaling from skeletal muscle cells to motor nerve terminals is a recognized mechanism for modulating the strength of neuromuscular transmission. We recently described a form of long-term depression of transmitter release at the mature neuromuscular junction that is dependent on the production of nitric oxide, most likely by the muscle cell (Etherington and Everett 2004 J Physiol (Lond) 559:507-517). We now show that the depression is blocked by treating neuromuscular preparations with mu-conotoxin G111A, an antagonist of skeletal muscle voltage gated sodium channels, indicating that the depression requires postsynaptic action potential firing. Experiments on dually-innervated sartorius muscles revealed that propagation of action potentials generated by low-frequency stimulation of one nerve branch gives rise to nitric-oxide mediated depression at unstimulated nerve terminals located many millimetres away on the same muscle fiber. The non-Hebbian pattern of expression of the depression, as well as its reliance on postsynaptic action potential firing, distinguish it from forms of synaptic depression described at immature neuromuscular synapses and may provide a mechanism for coregulation of the strength of motoneurons innervating the same postsynaptic cell.

Distinct activities of novel neurotoxins from Australian venomous snakes for nicotinic acetylcholine receptors.

Envenomation from Australian elapid snakes results in a myriad of neurological effects due to post-synaptic neurotoxins that bind and inhibit nicotinic acetylcholine receptors (nAChRs) of neurons and muscle fibres. However, despite the significant physiological effects of these toxins, they have remained largely undercharacterised at the molecular level. This study describes the identification and comparative analysis of multiple neurotoxin isoforms from ten Australian snakes, including functional characterisation of two of these isoforms, Os SNTX-1 from Oxyuranus scutellatus and the more potent Pt LNTX-1 from Pseudonaja textilis. Electrophysiological recordings from adrenal chromaffin cells demonstrate that both neurotoxins act as competitive antagonists of nAChRs in a concentration-dependent manner. Their effects upon spontaneous and nerve-evoked membrane responses at the amphibian neuromuscular junction provide further evidence that both toxins bind muscle nAChRs in an irreversible manner. This study represents one of the most comprehensive descriptions to date of the sequences and activity of individual Australian elapid neurotoxins.

Mechanisms of calcium sequestration during facilitation at active zones of an amphibian neuromuscular junction

The calcium transients ( Δ [ Ca 2 + ] i ) at active zones of amphibian ( Bufo marinus) motor-nerve terminals that accompany impulses, visualized using a low-affinity calcium indicator injected into the terminal, are described and the pathways of subsequent sequestration of the residual calcium determined, allowing development of a quantitative model of the sequestering processes. Blocking the endoplasmic reticulum calcium pump with thapsigargin did not affect Δ [ Ca 2 + ] i for a single impulse but increased its amplitude during short trains. Blocking the uptake of calcium by mitochondria with CCCP had little effect on Δ [ Ca 2 + ] i of a single impulse but greatly increased its amplitude during short trains. This present compartmental model is compatible with our previous Monte Carlo diffusion model of Ca 2 + sequestration during facilitation [Bennett, M.R., Farnell, L., Gibson, W.G., 2004. The facilitated probability of quantal secretion within an array of calcium channels of an active zone at the amphibian neuromuscular junction. Biophys. J. 86(5), 2674–2690], with the single plasmalemma pump in that model now replaced by separate pumps for the plasmalemma and endoplasmic reticulum, as well as the introduction of a mitochondrial uniporter.

Modulation of Calcium-Dependent and -Independent Acetylcholine Release From Motor Nerve Endings

Inhibition of acetylcholine (ACh) release by adenosine is an important mechanism by which the secretory apparatus is regulated at both mammalian (Ginsborg and Hirst, 1972; Hirsh et al., 2002; Silinsky, 2004) and amphibian (Silinsky, 1980; Silinsky and Solsona, 1992; Redman and Silinsky, 1993, 1994; Robitaille et al., 1999) neuromuscular junctions (NMJs). ACh is known to be costored with ATP in cholinergic vesicles (Zimmermann, 1994), and it has been demonstrated that at amphibian NMJs, adenosine derived from neurally released ATPis the mediator of neuromuscular depression exhibited at low frequencies of nerve stimulation (Redman and Silinsky, 1994) (Fig. 1). At the mouse motor nerve ending the inhibitory actions of adenosine on transmitter release are linked to a reduction in the nerve-terminal calcium current associated with neurotransmitter release (Silinsky, 2004). In contrast, at the frog motor nerve, inhibition of ACh release by adenosine occurs in the absence of any effect on nerve-terminal calcium currents (Silinsky and Solsona, 1992; Redman and Silinsky, 1994; Robitaille et al., 1999). That is, at the frog NMJ adenosine inhibits ACh release through an effect on a process that takes place downstream from calcium entry. Although the exact site at which adenosine inhibits transmitter release is unknown, both the speed (50-100 ms; E. M. Silinsky, unpublished observations) and the stimulation-independent nature of inhibition suggest that this process must occur through an action on vesicles that are already primed and ready for release. Thus, the likely sites for mediating the action of adenosine are those core components of the neurotransmitter release process, the three SNARES (SNAP-25, syntaxin, and synaptobrevin), and synaptotagmin. However, there are difficulties in addressing which of these individual elements of the secretory apparatus might be involved in the actions of adenosine. We could use fractions of botulinum toxin to eliminate individual components of the secretory apparatus. However, each of these core components of the release machinery is individually essential for the neurotransmitter release process. Therefore, we decided to approach this problem by alternative means.

The Facilitated Probability of Quantal Secretion within an Array of Calcium Channels of an Active Zone at the Amphibian Neuromuscular Junction

A Monte Carlo analysis has been made of the phenomenon of facilitation, whereby a conditioning impulse leaves nerve terminals in a state of heightened release of quanta by a subsequent test impulse, this state persisting for periods of hundreds of milliseconds. It is shown that a quantitative account of facilitation at the amphibian neuromuscular junction can be given if the exocytosis is triggered by the combined action of a low-affinity calcium-binding molecule at the site of exocytosis and a high-affinity calcium-binding molecule some distance away. The kinetic properties and spatial distribution of these molecules at the amphibian neuromuscular junction are arrived at by considering the appropriate values that the relevant parameters must take to successfully account for the experimentally observed amplitude and time course of decline of F1 and F2 facilitation after a conditioning impulse, as well as the growth of facilitation during short trains of impulses. This model of facilitation correctly predicts the effects on facilitation of exogenous buffers such as BAPTA during short trains of impulses. In addition, it accounts for the relative invariance of the kinetics of quantal release due to test-conditioning sequences of impulses as well as due to change in the extent of calcium influx during an impulse.

Phorbol esters and adenosine affect the readily releasable neurotransmitter pool by different mechanisms at amphibian motor nerve endings.

Phorbol esters and adenosine have been proposed to interact at common sites downstream of calcium entry at amphibian motor nerve endings. We thus studied the actions and interactions of phorbol esters and adenosine using electrophysiological recording techniques in conjunction with both binomial statistical analysis and high-frequency stimulation at the amphibian neuromuscular junction. To begin this study, we confirmed previous observations that synchronous evoked acetylcholine (ACh) release (reflected as endplate potentials, EPPs) is well described by a simple binomial distribution. We then used binomial analysis to study the effects of the phorbol ester phorbol dibutyrate (PDBu, 100 nM) and adenosine (50 microM) on the binomial parameters n (the number of calcium charged ACh quanta available for release) and p (the average probability of release), where the mean level of evoked ACh release (m) = np. We found that PDBu increased m by increasing the parameter n whilst adenosine reduced m by reducing n; neither agent affected the parameter p. PDBu had no effect on either the potency or efficacy of the inhibition produced by adenosine. Subtle differences between these two agents were revealed by the patterns of EPPs evoked by high-frequency trains of stimuli. Phorbol esters increased ACh release during the early phase of stimulation but not during the subsequent plateau phase. The inhibitory effect of adenosine was maximal at the beginning of the train and was still present with reduced efficacy during the plateau phase. When taken together with previous findings, these present results suggest that phorbol esters increase the immediately available store of synaptic vesicles by increasing the number of primed vesicles whilst adenosine acts at a later stage of the secretory process to decrease the number of calcium-charged primed vesicles.

The formation and function of single transmitter release sites at mature amphibian motor-nerve terminals.

The extent of quantal transmitter release from single sites of synaptic vesicle accumulations along the length of motor-nerve terminal branches at the amphibian neuromuscular junction has been investigated. Such a determination involves development of a model for the generation of quantal potential fields at single styryl-dye stained sites along the length of a branch. Successful testing and application of this model indicates that the extent of quantal release at a dye-stained site is proportional to the total length of active zone at the site. The stability of these sites and of their ensheathing terminal Schwann cell processes was also investigated. Following simultaneous injection of the terminal Schwann cell and nerve terminal with different fluorescent dyes, terminal branches were observed to show dynamic changes in their length, with these occurring in relatively short periods of hours or less. Redistribution of styryl dye stained sites at the ends of branches also occurred in such short periods of time. These were accompanied by changes in the configuration of terminal Schwann cells, which generally occurred prior to changes in the length of nerve terminal branches.

Calcium Channel Isoforms Underlying Synaptic Transmission at EmbryonicXenopusNeuromuscular Junctions

Studies on the amphibian neuromuscular junction have indicated that N-type calcium channels are the sole mediators of stimulus-evoked neurotransmitter release. We show, via both presynaptic and postsynaptic voltage-clamp measurements, that dihydropyridine (DHP)-sensitive calcium channels also contribute to stimulus-evoked release at developing Xenopus neuromuscular junctions. Whereas inhibition of postsynaptic responses by omega-conotoxin (omega-Ctx) GVIA has been taken previously as evidence that only N-type channels mediate transmitter release, we find that both N-type and DHP-sensitive calcium currents are sensitive to this toxin. The unusual sensitivity of DHP-sensitive calcium channels to omega-Ctx GVIA in presynaptic terminals raises the possibility that this channel type may have escaped detection in previous physiological studies on adult frog neuromuscular junctions. Alternatively, the additional channel isoforms may be present only during early development, when they may serve to strengthen collectively presynaptic release during critical periods of synaptogenesis.

ERRATUM: Everett A.W. 1999. Membrane Recycling Due to Low and High Rates of Nerve Stimulation at Release Sites in the Amphibian (Bufo marinus) Neuromuscular Junction. Synapse 32:110-118.

ERRATUM: Everett A.W. 1999. Membrane Recycling Due to Low and High Rates of Nerve Stimulation at Release Sites in the Amphibian (Bufo marinus) Neuromuscular Junction. Synapse 32:110-118. In the above-referenced article, Figure 1 was erroneously reproduced in black and white as converted from a color original, not providing an accurate representation of the staining. The figure is reproduced here as the author intended, from an original black and white rendering. The Publisher sincerely apologizes for this error.

Membrane recycling due to low and high rates of nerve stimulation at release sites in the amphibian (Bufo marinus) neuromuscular junction

Membrane recycling due to low and high rates of nerve stimulation at release sites in the amphibian (Bufo marinus) neuromuscular junction

Membrane recycling due to low and high rates of nerve stimulation at release sites in the amphibian (Bufo marinus) neuromuscular junction.

The activity-dependent labelling of motor nerve terminals with the dye FM1-43 has been used to estimate the relative levels of membrane recycling (due to synaptic vesicle exocytosis and recovery) at release sites in response to 1,200 nerve stimulations delivered at either low (0.5 Hz) or high (30 Hz) frequency. Dye in terminals appears as fluorescent spots distributed along the terminal branches; each spot is thought to be a cluster of labelled vesicles associated with a release site. Relative fluorescence in spots was quantified from images obtained with a confocal microscope. Spot intensities varied widely within branches following labelling at both frequencies, but the distribution was highly skewed towards lower intensities at low frequency stimulation; at high frequency, more spots had stronger fluorescence. Both weak and strongly stained spots were uniformly distributed along the length of terminal branches after low frequency stimulation; however, there was a gradual decline in all spot intensities towards the distal end of branches loaded with dye at high frequency stimulation. Antibody staining for synaptic vesicles was, on average, uniformly distributed along the branches. The increase in number of more strongly FM1-43-labelled spots in terminal branches stimulated at high compared with low frequency suggests that more release sites are active at high rates of nerve stimulation. This "recruitment" of release sites at high frequency stimulation occurs mostly in the proximal half of terminal branches and is not related to the abundance of synaptic vesicles in the terminal.

Endogenous peptidergic modulation of perisynaptic Schwann cells at the frog neuromuscular junction.

1. Although peptides are important modulators of synapses, their action on synapse-glia interactions remain unclear. The amphibian neuromuscular junction (NMJ) was used to examine the effects of substance P (SP) on perisynaptic Schwann cells (PSCs), glial cells at the frog NMJ, by monitoring changes in intracellular Ca2+. 2. SP induced Ca2+ responses that were mimicked by the neurokinin 1 receptor (NK-1) agonist septide and with a shorter delay by the SP fragment, SP(6-11). SP and SP(6-11) responses were blocked by NK-1 antagonists SR140333 and LY303870. 3. Ca2+ responses remained unchanged when extracellular Ca2+ was removed but were blocked after pertussis toxin (PTX) treatment, indicating that the receptors were linked to internal stores of Ca2+ via a PTX-sensitive G-protein. 4. The slowly hydrolysable NK-1 agonist [Sar9, Met(O2)11]-SP only induced Ca2+ responses when applied for a long period of time and not during brief, local applications, suggesting the involvement of SP hydrolysis. Acetylcholinesterase (AChE) may not be involved in SP degradation since Ca2+ responses evoked by SP were unchanged in the presence of the cholinesterase inhibitor neostigmine. 5. Ca2+ responses induced by muscarine and nerve stimulations were almost abolished when preceded by SP applications, while those induced by ATP were significantly reduced. The rundown of the nerve-evoked Ca2+ responses in PSCs was attenuated in the presence of SR140333. 6. These results indicate that endogenous SP is involved in the regulation of PSC activity and that SP is an important modulator of glial cell Ca2+ signalling and synapse-glia communication.

A single site on the ϵ subunit is responsible for the change in ACh receptor channel conductance during skeletal muscle development

Four critically positioned amino acids on each of the αβ, δ and γ sunits of the Torpedo nicotinic acetylcholine receptor are determinants of channel conductance. Our results show that the γ and ϵ subunits of Xenopus muscle receptors are identical at all four positions, despite the fact that α 2 βδϵ receptors have a 50% greater conductance than α 2 βδγ receptors. Instead, the functional difference is conferred by a single charged residue that lies extracellular to all four positions, corresponding to a location in the Torpedo receptor previously shown to have no influence on conductance. Substitution of a positively charged lysine residue in γ by the neutral methionine in ϵ at this extracellular position is responsible for the increased conductance during maturation of the amphibian neuromuscular junction.