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. 2011 Jan 4;589(Pt 5):1133–1142. doi: 10.1113/jphysiol.2010.202994

Microtransplantation of acetylcholine receptors from normal or denervated rat skeletal muscles to frog oocytes

Annalisa Bernareggi 1, Jorge Mauricio Reyes-Ruiz 2, Paola Lorenzon 1, Fabio Ruzzier 1, Ricardo Miledi 2,3
PMCID: PMC3060592  PMID: 21224230

Abstract

Cell membranes, carrying neurotransmitter receptors and ion channels, can be ‘microtransplanted’ into frog oocytes. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have, still embedded in their natural lipid environment. This approach has been previously demonstrated to be very useful to study neurotransmitter receptors and ion channels contained in cell membranes isolated from human brains. Here, we examined the possibility of using the microtransplantation method to study acetylcholine receptors from normal and denervated rat skeletal muscles. We found that the muscle membranes, carrying their fetal or adult acetylcholine receptor isoforms, could be efficiently microtransplanted to the oocyte membrane, making the oocytes become sensitive to acetylcholine. These results show that oocytes injected with skeletal muscle membranes efficiently incorporate functional acetylcholine receptors, thus making the microtransplantation approach a valuable tool to further investigate receptors and ion channels of human muscle diseases.

Non-technical summary

Nicotinic acetylcholine receptors (nAChRs) expressed by skeletal muscle are the key proteins of the motoneuron–muscle communication necessary to induce muscle contraction. The biophysical and pharmacological characterization of the receptors in adult human skeletal muscle is limited by the difficulties associated with obtaining, and maintaining, suitable biopsy material. In this paper, we characterized some nAChR properties of innervated and denervated skeletal muscle by injecting the membranes into Xenopus oocytes. Such an approach, termed the microtransplantation technique, offers two main advantages: (1) direct characterization of the original receptors, still embedded in their natural lipid environment with their associated molecules; and (2) the possibility of using membranes isolated from postmortem frozen tissues. We demonstrate that this technique can be a very simple and useful approach to study skeletal muscle receptors and ion channels under different physiological and pathological conditions.

Introduction

Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane proteins that form cation-selective ion channels (reviewed by Changeux & Edelstein, 2005). There are dozens of nAChRs subtypes, made up of specific combinations of 17 different subunits. These receptors are involved in fast excitatory neurotransmission at the neuromuscular junction, and at synapses in the central and peripheral nervous systems. In skeletal muscle, the expression and localization of nAChRs change during muscle development (Miledi, 1959; Diamond & Miledi, 1962). The fetal isoform ((α1)2, β1, γ, δ) is expressed in myoblasts and myotubes, where the receptors are diffusely distributed along the entire cell surface. After innervation, the fetal isoform is progressively replaced by the adult isoform where the γ subunit is substituted by the ɛ subunit (Mishina et al. 1986) and the receptors become localized to the neuromuscular junctional and juxta-synaptic regions of skeletal muscle fibres (Miledi, 1960b). It is also known that after denervation, the fetal nAChR subunit isoform is re-expressed, and the receptors spread outside the endplate region (Axelsson & Thesfleff, 1959; Miledi, 1960a; Miledi & Trowell, 1962), similarly to what is observed in some human neuromuscular disorders (Gattenlöhner et al. 2002; Zhu et al. 2006).

The study of human muscle diseases is severely limited by the difficulties associated with obtaining, and maintaining, suitable biopsy material. For many years, injection of skeletal muscle mRNAs and cRNAs into Xenopus oocytes has been used as a model to study ion channels (Miledi et al. 1982, 1989; Parker et al. 1988). With this method, the DNAs are transcribed, translated, processed and assembled into the plasma membrane by the oocyte's own protein synthesizing machinery. Therefore, the native receptors in their original cells may have a different cohort of associated proteins and lipids that could confer to native receptors properties different from those of the receptors expressed by the oocytes. Some years ago, the microtransplantation technique (Marsal et al. 1995) was used as an alternative approach to study neurotransmitter receptors in neurological diseases (Palma et al. 2002; Miledi et al. 2004; Bernareggi et al. 2007; Limon et al. 2008; reviewed by Miledi et al. 2006). Two important advantages of this technique are that: (1) it allows a direct characterization of the original receptors, with their associated molecules and still embedded in their natural lipid environment; and (2) it allows studies of receptors from postmortem frozen tissues.

In this paper we investigate the possibility of using the microtransplantation technique as a new approach to study receptors and ion channels from skeletal muscles. For that purpose, we determined the capability of Xenopus oocytes to incorporate acetylcholine receptors from normal and denervated skeletal muscles of the rat.

Methods

Ethical approval

Animal care and treatment were conducted in conformity with institutional guidelines in compliance with national and international laws and policies (European Economic Community (EEC) Council Directive 86/609; OJL 358; December 12, 1987) and under the guidelines of the IACUC protocol 2006-2682 USA.

Adult Wistar rats (n= 12) weighing approximately 300 g were anaesthetised with a mixture of ketamine (90 mg kg−1) and xylazine (10 mg kg−1). Muscle denervation was performed by transecting the sciatic nerve of the right leg (about 5 cm from the nerve ending). The animals were killed by CO2 and the gastrocnemius, soleus and tibialis anticus muscles were collected at 1, 2 and 4 weeks after denervation. The contralateral (left leg) muscles were used as controls. Dissected tissues were placed immediately in liquid nitrogen and stored at −80°C until processed.

Twenty adult female Xenopus laevis frogs were fully anaesthetized by immersion in cold 0.17% MS-222 for 15 min and the pieces of ovary were aseptically removed according to the protocol described by Miledi et al. (2006). At the end of the experiments animals were killed by prolonged anaesthesia.

RNA preparation

Muscle samples (0.6–1 g) were homogenized and total RNA was extracted using the Trizol method (Invitrogen, Carlsbad, CA, USA). The integrity of RNA was assessed by gel electrophoresis and its concentration was measured by spectrophotometry. Messenger RNAs were isolated using Oligotex resin (Qiagen, Hilden, Germany).

Real-time quantitative PCR (qRT-PCR)

mRNA (2 μg) was used as template to obtain cDNA with Bio-Rad's iScript and then used to perform the qRT-PCR in triplicate using the SYBR green mix from Bio-Rad and an iCycler RT-PCR machine. For data analysis, threshold cycles obtained during the real-time PCR were analysed with the Inline graphic method (Livak & Schmittgen, 2001), which can be used to calculate the fold change in gene expression normalized to an endogenous reference gene.

Membrane preparation

Skeletal muscle membranes were isolated from 0.5–1 g of frozen tissue according to the protocol described by Saito et al. (1984). The membranes (crude microsomes) were suspended in sterile water and kept frozen at −80°C until oocyte injection. Normal and denervated muscle samples were injected into oocytes of the same batch, in order to reduce variations due to donor frog, and other differences. Xenopus laevis follicles were dissected from segments of ovary, defolliculated with collagenase (0.5 mg ml−1, 30–40 min, Type I, Sigma, St Louis, MO, USA) and maintained at 16°C in Barth's solution (containing 100 units ml−1 of penicillin/streptomycin or gentamicin (0.5 mg ml−1, Sigma)). The next day, each membrane preparation was injected into oocytes at a protein concentration of 0.5–1 mg ml−1 (50 nl volume). Membranes were microinjected always into the animal pole of the Xenopus oocyte in close proximity to the equatorial band (for more details see Miledi et al. 2006).

Electrophysiology

One to four days after injection, ACh currents and Tout currents were recorded from voltage-clamped oocytes, using two microelectrodes filled with 3 m KCl (Miledi, 1982) and with the membrane potential held at −80 mV. The oocytes were superfused continuously at room temperature with Ringer solution (115 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 5 mm Hepes, adjusted to pH 7 with NaOH) in a purpose-designed recording chamber (RC-3Z, Warner Instruments, Hamden, CT, USA). Data acquisition and analyses was performed using WinWCP version 3.5 Strathclyde Electrophysiology software (kindly provided by John Dempster, Glasgow, UK).

All the drugs (Sigma) were applied using a constant perfusion system (5–10 ml min−1, VC-8 perfusion system, Warner Instruments). The flux speed was routinely controlled and maintained constant during each set of experiments. To guarantee a homogeneous and comparable cell perfusion, all the electrophysiological recordings were carried out on oocytes similarly oriented. Oocytes were placed in the recording chamber with the pole of the microinjected animal hemisphere facing toward the inlet port of the perfusion system.

For acetylcholine dose/current–response curves, the acetylcholine was repeatedly applied at 5 min intervals, and the half-dissociation constants (EC50) and Hill coefficients (nh) were estimated by fitting the data to Hill equations.

The time constant of ACh current decay (τi) was calculated by fitting the current decay to equation:

graphic file with name tjp0589-1133-m1.jpg

where I is the current, t is time and i is the exponential numbers.

To obtain I–V relations, the ACh current was normalized to the currents obtained at −160 mV. All values are expressed as means ±s.e.m. To calculate statistical significance, Student's t test was used and differences considered significant when P < 0.05.

Results

nAChRs in oocytes injected with rat skeletal muscle membranes

Membranes isolated from muscles were injected into the oocytes and these were then tested for responses to ACh. For simplicity, in here we shall call ‘innervated oocytes’ and ‘denervated oocytes’ those oocytes that were injected with membranes isolated from innervated or denervated muscles, respectively. Membrane current recordings showed that 24 h after the injection the innervated and denervated oocytes had already incorporated many functional nAChRs and thus became sensitive to ACh. Since mRNAs are easily degraded, and because the protocol used to isolate the membranes does not take precautions to prevent that degradation, it is very unlikely that the ACh-induced currents recorded in the oocytes were due to the expression of receptors synthesized by the oocytes from exogenous mRNAs.

Sample ACh currents, obtained during the first applications of ACh (1 mm), recorded from oocytes injected with membranes of innervated, 1 week-, 2 weeks- and 4 weeks-denervated rat skeletal muscles are illustrated in Fig. 1A. It is known that occasionally Xenopus oocytes have native muscarinic AChRs (Kusano et al. 1982; Miledi et al. 1989), but the oocytes have never been found to clearly possess endogenous nAChRs (R. Miledi, unpublished observation). In the present experiments, only two batches of non-injected oocytes elicited small muscarinic responses to ACh; for those oocytes we used atropine (1 μm) in the perfusing solution to avoid activating the endogenous muscarinic receptors.

Figure 1. ACh currents of oocytes injected with membranes from innervated or denervated muscles.

Figure 1

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A, sample currents evoked by ACh (1 mm) in oocytes injected 1 day previously with membranes from innervated, or 1, 2 and 4 weeks rat denervated muscles (1w, 2w and 4w). Holding potential −80 mV. B, mean ACh currents at 1 day post-injection (5–6 oocytes, 1 frog and 1 membrane preparation for each column). C, the current amplitude was maximal after 1 day post-injection and then decreased. Oocytes from the same frog; 5–6 oocytes for each value. All the currents are those induced by the first application of ACh. Error bars are s.e.m.

It is well known that denervation causes skeletal muscles to become ‘supersensitive’ to ACh, because of the appearance of many new nAChRs in the extrajunctional regions of the muscle fibres (Axelsson & Thesfleff, 1959; Miledi, 1959, 1960a; Miledi & Potter, 1971). Accordingly, the ACh current amplitudes generated by the oocytes injected with denervated muscle membranes were increased. Namely, while in innervated muscle oocytes the currents generated by 1 mm ACh were only a few nanoamps (ranging from 2 nA to about 20 nA), in denervated muscle oocytes the ACh current amplitudes were significantly higher, and ranged from 36 nA (1 week-denervated, 3 days post-injection) to about 1 μA (4 week-denervated, 1 day post-injection, data not shown). Moreover, many innervated oocytes failed to elicit ACh currents: the ACh-responsive innervated oocytes were only about 10% (14 membrane preparations, n= 54) while 100% of the denervated oocytes responded to ACh (9 membrane preparations, 3 for each denervation period, n > 50).

Already 24 h after the injection, the amplitude of ACh currents recorded from 1 week-denervated oocytes was appreciably greater than that elicited by oocytes injected with innervated muscle membranes, and the difference became greater after 2 and 4 week denervation (Fig. 1B). Furthermore, in all cases the ACh currents were already maximal at 1 day post-injection and decreased during the following days (Fig. 1C). All values reported in Fig. 1 correspond to currents elicited by the first applications of ACh.

Voltage-gated Ca2+ channels from innervated or denervated muscles are incorporated stably into oocyte membrane

A set of experiments was performed to explore the incorporation of other ion channels. In particular, we assessed the integration of voltage-operated Ca2+ channels, which can be easily monitored measuring the Tout currents in injected oocytes. The Tout current (Ca2+-dependent transient chloride outward current) is a chloride current, caused by an increase of intracellular Ca2+ ions that follows the opening of voltage-gated Ca2+ channels, that is selectively blocked by Mn2+ ions. Thus, the measurement of Tout current is a simple and convenient method to monitor the presence of Ca2+ channels in the oocyte membrane (Miledi, 1982).

Xenopus oocytes have native voltage-gated Ca2+ channels that open when the membrane is depolarized (Miledi, 1982). The Tout current of oocytes injected with either innervated or denervated skeletal muscle membranes was significantly greater than the native Tout current of non-injected oocytes (Fig. 2A and B), showing that the injected oocytes incorporated functional voltage-operated Ca2+ channels present in the muscle membranes. Interestingly, the Tout current did not decrease during the three post-injection days examined. These results indicated that voltage-gated Ca2+ channels remained incorporated more stably than nAChRs, whose currents diminished (cf. Fig. 2C and Fig. 1C).

Figure 2. Microtransplantation of Ca2+ channels from innervated or denervated muscles to oocytes.

Figure 2

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A, currents elicited by a 3 s depolarising step from the holding potential of −80 mV to +20 mV in a non-injected oocyte and in an innervated oocyte (2 days post-injection). B, amplitude of Tout currents (current blocked by 5 mm Mn2+) after 1 day post-injection. C, Tout current amplitudes at different post-injection days (n= 3–6 oocytes each, oocytes from the same frog).

ACh current decay in innervated and denervated muscle oocytes

Prolonged applications of ACh (1 mm for 60–90 s) to denervated muscle oocytes gave currents characterized by more than one time constant of decay (Fig. 3A, upper trace). The best fit to such decay was obtained using a triple-exponential function (τf, fast decay constant; τs1 and τs2, slow decay constants). Prolonging agonist application up to 40 s, the ACh current decay became again more rapid in all denervated oocytes.

Figure 3. Desensitization of ACh currents in innervated and denervated muscle oocytes.

Figure 3

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A, the ACh current decays in denervated muscle oocytes were well fitted by a triple-exponential equation (upper trace, 1 week (1w)-denervated oocyte), while the ACh currents of innervated oocytes were best fitted by a double-exponential equation (bottom trace, innervated oocyte). B, comparison of decay time constants (τf, τs1) of innervated and denervated muscle oocytes.

In contrast, the ACh current in innervated muscle oocytes was best fitted by a double-exponential function (τf and τs1; Fig. 3A, bottom trace). The values of τf were found to be similar in innervated (0.52 ± 0.02 s, n= 3) and denervated oocytes (0.56 ± 0.03 s, n= 10), whereas τs1 values were slightly different: 3.86 ± 0.20 s, n= 3 vs. 6.14 ± 0.33 s, n= 10, innervated and denervated, respectively (Fig. 3B). The τs2 value for denervated oocytes was 16.11 ± 3.33 s (n= 10). Therefore, the overall desensitization of the ACh current in innervated muscle oocytes was faster than in denervated muscle oocytes.

ACh current ‘run-down’ in denervated muscle oocytes

As the ACh current amplitude in innervated muscle oocytes was only a few nanoamps, we did not analyse the properties of those transplanted nAChRs in more detail and concentrated in determining some properties of the Ach currents recorded in denervated muscle oocytes.

Repetitive applications of 1 mm ACh to denervated oocytes generated currents of decreasing amplitudes (Fig. 4A). Such ACh concentration resembles the local concentration of the transmitter at the neuromuscular junction (Land et al. 1981), which activates the entire receptor population at the junction (Dudel et al. 1992). The agonist was applied for 10 s and washed out before complete receptor densensitization. The records frequently show a ‘wash-kick’ (Fig. 4A), probably due to open-channel blockage by high concentrations of ACh (Odgen & Colquhoun, 1985).

Figure 4. Recovery of ACh currents in oocytes injected with denervated muscle membranes.

Figure 4

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A, representative currents induced by the 1st, 2nd and 6th of consecutive ACh applications (1 mm, 0, 4 and 20 min, holding −80 mV) in an oocyte, 1 day post-injection of 2 week (2w)-denervated muscle membranes. The decrease of current induced by repetitive ACh applications (1 mm for 10 s, time interval 4 min) was observed with all the samples tested (B) and at different times after injection, 1 week denervated muscles membranes (C). 3–6 oocytes were analysed for each experimental point.

The ACh current ‘run-down’ was monitored by applying ACh (1 mm) at intervals of 4 min and measuring the peak amplitudes of the ACh currents evoked. In spite of a large variability in the run-downs obtained with different muscle samples, different donor frogs and membrane preparations, the ACh currents always decreased during repetitive applications (Fig. 4B) and the fall was preserved independently from the time after injection (Fig. 4C).

To test the effect of agonist concentration on the ACh current ‘run-down’, we analysed currents induced by 100 μm and 10 μm ACh applied at 5 min intervals to 1 week-denervated oocytes. The ACh currents induced by 100 μm always decreased in amplitude and recovered only slightly after more than 0.5 h following removal of the ACh (25.55 ± 13.74% of the first application, n= 3, Fig. 5A). In the presence of a lower agonist concentration (10 μm), the run-down was significantly reduced but not completely abolished (6th application, 86.77 ± 7.6%n= 11, Fig. 5B).

Figure 5. ACh current run-down.

Figure 5

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A, left, ACh (100 μm) currents at the first and 6th ACh applications in a 1 week (1w)-denervated oocyte; right, averaged ACh current amplitudes during repetitive applications of ACh (100 μm) to oocytes injected with 1 week-denervated muscle membranes (n= 3). The ACh current falls to 13.47 ± 2.63% of the first current. Note, that after a washout of 35 min, the current recovered slightly to 25.55 ± 13.74% of the 1st current amplitude. B, left, ACh currents at the 1st and 6th applications of 10 μm ACh to a 1 week-denervated oocyte. The current amplitude at the 6th application decreased only to 86.77 ± 7.6% (n= 11, right). The ACh current run-down was dependent on the duration of the exposure to ACh. When ACh (100 μm) was applied for 2 s (C, left) the run-down was not noticed (at the 6th application the current was 131 ± 16.2%, n= 3, right). All the results shown here were performed in oocytes isolated from the same frog.

The duration of the exposure to ACh also significantly affected the current ‘run-down’. When 100 μm ACh was applied for 2 s the run-down was not observed (n= 3) and at the 6th ACh application the current was even increased to 131 ± 16.2% (n= 3) of the 1st ACh application (Fig. 5C).

Pharmacology of microtransplanted nAChRs

ACh current–voltage (I–V) relations in denervated muscle oocytes showed a reversal potential close to 0 mV, as well as typical current rectification (Methfessel et al. 1986) (Fig. 6A, n= 5). The ACh dose–current response relation gave an EC50 of 20 ± 1.4 μm and nH of 2.2 ± 0.25 (n= 4, Fig. 6B).

Figure 6. Properties of denervated muscle nAChRs microtransplanted to oocytes.

Figure 6

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A, I–V relation of oocytes injected with denervated muscle membranes (n= 5, 2 frogs). Currents were induced by 10 μm ACh and normalized to those recorded at −160 mV. The reversal potential was near 0 mV. B, ACh dose–response relation obtained in oocytes injected with membranes of denervated muscle (see text for further details; 1 frog, n= 4). C, blockage of ACh currents (10 μm) by dTC in an oocyte injected with 1 week-denervated muscle membranes. D, representative ACh current recorded in another oocyte injected with the same preparation and blockage after 5 min incubation with α-bungarotoxin (α-BuTX).

d-Tubocurarine (dTC), a competitive and reversible antagonist of muscle nAChRs (Wenningmann & Dilger, 2001), blocked the ACh (10 μm) current with an IC50 of 31.45 ± 8.44 nm (n= 5), and the ACh current recovered completely after 10 min of washout (not shown). The effect of two different dTC concentrations on ACh (10 μm) currents induced in 1 week-denervated injected oocytes is illustrated in Fig. 6C.

The microtransplanted nAChRs are irreversibly blocked by α-bungarotoxin (α-BuTX). Figure 6D illustrates the currents elicited by 10 μm ACh in an oocyte injected with 1 week-denervated skeletal muscle, in the absence and in the presence of the toxin. After 5 min incubation with 5 μmα-BuTX, the ACh current was completely blocked (n= 4). To test the effect of dTC and α-BuTX, a low agonist concentration (10 μm) was used to avoid the ACh-induced current run-down.

Quantitative RT-PCR (qRT-PCR) of nAChR subunits

The same tissues used for obtaining the muscle membranes were used for qRT-PCR experiments. We monitored the expression of several nAChRs subunits in rat skeletal muscle. In particular, we compared the levels of α1, β1, γ, δ and ɛ subunits in the innervated and 1, 2 and 4 weeks-denervated muscles. All subunits displayed a similar pattern of upregulation and were already appreciably increased 1 week after denervation. As illustrated in Fig. 7, the level of expression of the β1, γ and δ subunits was well maintained from the 2nd to the 4th week, whilst that of the α1 and ɛ subunits appeared to decrease slightly.

Figure 7. Expression of nAChR subunits in rat muscles at 0, 1, 2 and 4 weeks after sciatic nerve transection.

Figure 7

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There is a large increase in the expression of nAChR subunits in the denervated muscles, reaching a maximum at about 2 weeks post-denervation. Average qRT-PCR from innervated or denervated leg muscles of two rats.

Discussion

Some skeletal muscle diseases are associated with defects in nAChRs and ion channels as well as with alterations in their expression and distribution (Vincent, 2006). For example, in amyotrophic lateral sclerosis there is a re-expression of the fetal isoform of nAChR, as a consequence of motor neuron dysfunction (Tsujihata et al. 2001). Furthermore, in some neurogenic and myogenic disorders, there is an upregulation of the nAChR γ subunit (Gattenlöhner et al. 2002; Zhu et al. 2006). Since one of the main hurdles in studying the functionality of receptors/channels from human skeletal muscles is the low availability of suitable biopsy material, we suggest that the microtransplantation technique is a convenient and potent alternative approach to the traditional methods.

It is well documented (e.g. Witzemann et al. 1990; Adams et al. 1995) that sectioning the motor axons results in an increased expression of the fetal isoform of nAChRs, especially during the first month of muscle denervation, a period that corresponds to that when the muscle can recover some of its function. We studied ACh currents of oocytes injected with cell membranes isolated from innervated muscles and muscles denervated for up to 4 weeks and, as expected (Miledi & Potter, 1971; Miledi et al. 1982), we found that oocytes injected with membranes derived from denervated muscles generated ACh currents that were more than 50 times greater (up to 1 μA) than those elicited in oocytes injected with membranes from the innervated muscles. Similar current amplitudes were reported by Parker et al. (1988), after injecting oocytes with nAChR mRNAs isolated from innervated and denervated skeletal muscles. This is not surprising: in normally innervated muscle fibres the nAChRs are mainly located at the end-plate region, while in denervated muscle fibres the receptors are spread in high numbers over their entire surface (Axelsson & Thesfleff, 1959; Miledi, 1959, 1960a). It seems that the increased number of nAChRs caused by muscle denervation is due mainly to an increased transduction of the genes encoding the various nAChR subunits (Fig. 7 and see also Witzemann et al. 1990; Adams et al. 1995). Our qRT-PCR analyses revealed an upregulation of the expression of all the nAChR subunits in denervated muscles, in agreement with the increased ACh currents of oocytes microtransplanted with membranes from denervated muscles.

We also observed that only about 10% of oocytes injected with membranes from innervated muscles responded to ACh compared with 100% for oocytes injected with membranes from denervated muscles. The evidence that innervated as well as denervated oocytes stably incorporated functional Ca2+ channels, as demonstrated by recordings of Tout currents, further excludes the possibility that the small number of ACh-responsive innervated oocytes was due to the ‘quality’ of membrane preparations. This confirms the reliability of our approach and suggests that the microtransplantation of endplate membranes will increase the efficiency of ‘junctional’ nAChR incorporation.

It is worth pointing out that, even if the aim of the present work was not the characterisation of Ca2+ channels, our results show that the microtransplantation technique can be used to study many other muscle receptors and channels. We noticed that oocytes injected with 4 week-denervated skeletal muscles incorporated more Ca2+ channels than those injected with innervated muscles (Fig. 2), in line with the observation that postsynaptic Ca2+ channels are generally over-expressed after denervation (Péréon et al. 1997).

Interestingly, the incorporation of functional nAChRs into the membrane of oocytes injected with denervated muscle membranes was maximal after about 1 day, and by the third day post-injection the ACh current had decreased to 20–50% (Fig. 1C). In contrast, the incorporation of Ca2+ channels, from the same membrane preparations, was still maintained (Fig. 2C). On the other hand, we have previously reported that oocytes injected with brain membranes continue to incorporate GABA and glutamate receptors for a few days after injection (Miledi et al. 2004; Bernareggi et al. 2007). This difference between brain and muscle membranes may be related to the different turnover of nAChRs observed in vivo: in adult fibres nAChRs are internalized and degraded slowly (t1/2= 10 days); after denervation the newly synthesized nAChRs degrade at a faster rate (t1/2= 1 day, Shyng & Salpeter, 1990). It may be that the membranes carrying nAChRs from denervated muscle do not remain for long in the oocyte plasma membrane.

The presence of the ɛ subunit in the adult isoform of muscle nAChRs is responsible for an ACh current desensitization that is faster than that of the fetal isoform, expressing the γ subunit (Morales & Sumikawa, 1992). We observed similar properties between the ACh currents of innervated and denervated oocytes, the latter characterised by longer slow decay constants (τs1 and τs2).

The ACh currents of denervated oocytes recovered completely from desensitization when the ACh (100 μm) was applied for a short period of time (2 s). This result is in line with what has been reported for transfected cells expressing the fetal isoform of nAChRs (Reitsetetter et al. 1999). However, prolonged applications of ACh to fetal nAChRs are followed by a slow recovery from desensitization. In cells transfected with the fetal isoform, Reitsetetter et al. (1999) reported full recovery from desensitization in about 4 min, after a 30 s application of ACh (1 mm). We observed that for application times of 10 s (100 μm or 1 mm), the ACh current elicited by denervated-injected oocytes did not recover fully, even after more than 35 min following the application of ACh. Since receptor desensitization is regulated by post-translational modifications such as glycosylation and phosphorylation (Huganir & Greengard, 1990), we suggest that the microtransplanted AChRs could retain some original characteristics that are missing in AChRs heterologously expressed from cDNA or mRNA. However, we cannot exclude that the run-down could also be due to internalization of the receptor induced by the agonist application, even if such an effect was reported in myotubes after a much longer treatment of different types of nAChR agonists (from 5 min up to 4 h, St. John & Gordon, 2001).

The current–voltage (I–V) relation of ACh currents of denervated oocytes had a reversal potential close to 0 mV and a current rectification typical of the nAChR muscle isoform (Methfessel et al. 1986). The currents showed an EC50 for ACh (20 ± 1.4 μm) similar to those reported for fetal nAChRs expressed by RNAs in oocytes (EC50= 26.6 μm, Purohit et al. 2007) or by myotubes in culture (EC50= 21.8 μm, Grassi et al. 1995). Moreover, one characteristic of acetylcholine activation is a positive cooperativity at the receptor binding sites that is responsible for a Hill coefficient of 1.3–3.0 (Jackson, 1989), which is in accord with the nH value obtained in our microtransplanted oocytes (nH= 2.2).

Among the competitive antagonists of the muscle isoform of nAChRs, dTC is able to reversibly bind the receptor, competing with the ACh binding sites (Arias, 1997). Our results show that 50% of the ACh current recorded in denervated oocytes was blocked by about 30 nm dTC, which is similar to the IC50 reported in the literature for the fetal isoform of the nAChR (41 nm, Wenningmann & Dilger, 2001). Finally, α-BuTX, the specific blocker of muscle nAChRs, completely and irreversibly blocked the microtransplanted nAChRs.

The central finding in this work is that, using the microtransplantation method, the original acetylcholine receptors and voltage-operated Ca2+ channels of innervated and denervated skeletal muscles can be studied well in Xenopus oocytes. Obviously, many more studies need to be done but we are convinced that the microtransplantation method will make it possible to study the receptors and channels from human muscles. This should allow us to understand better some human muscle diseases and thus help to develop better treatments.

Acknowledgments

This work was supported by grants from Progetto giovani ricercatori (University of Trieste, Italy) to A.B., MIUR-Italy (PRIN) to F.R. and P.L. and by the American Health Assistance Foundation to R.M. and J.M.R.-R. The authors are particularly grateful to Professor Pompeo Volpe (University of Padova, Italy) for helpful suggestions and to Francesco Fazzari for electrophysiological recordings. This paper is dedicated to our great friend Fabio Ruzzier.

Glossary

Abbreviations

α-BuTX

α-bungarotoxin

denervated (innervated) oocytes

oocytes injected with denervated (innervated) skeletal muscle

dTC

d-tubocurarine

Tout

Ca2+-dependent transient chloride outward current

Author contributions

All authors contributed to conception, design, analysis and interpretation of data, drafting the article and revising it critically. Electrophysiological experiments as well as membrane preparations were done at the University of Trieste and at the University of California Irvine. All authors approved the version to be published.

References

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