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• W2170918734 abstract "The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse. The efficiency of synaptic transmission between nerve and muscle depends on the number and density of acetylcholinesterase molecules (AChE) at the neuromuscular junction. However, little is known about the way this density is maintained and regulated in vivo. By using time lapse and quantitative fluorescence imaging assays in living mice, we demonstrated that insertion of new AChEs occurs within hours of saturating pre-existing AChEs with fasciculin2, a snake toxin that selectively labels AChE. In the absence of muscle postsynaptic activity or evoked nerve presynaptic neurotransmitter release, AChE insertion was decreased significantly, whereas direct stimulation of the muscle completely restored AChE insertion to control levels. This activity-dependent AChE insertion is mediated by intracellular calcium. In muscle stimulated in the presence of a Ca2+ channel blocker or calcium-permeable Ca2+ chelator, AChE insertion into synapses was significantly decreased, whereas ryanodine or ionophore A12387 treatment of blocked and unstimulated synapses significantly increased AChE insertion. These results demonstrated that synaptic activity is critical for AChE insertion and indicated that a rise in intracellular calcium either through voltage-gated calcium channels or from intracellular stores is critical for proper AChE insertion into the adult synapse. The maintenance of a high density of postsynaptic neurotransmitter receptors and transmitter inactivation molecules at the site of synaptic contact is critical for the functioning nervous system. In cholinergic synapses, acetylcholinesterase (AChE) 1The abbreviations used are: AChE, acetylcholinesterase; AChR, acetylcholine receptor; ColQ, collagen Q; α-BTX, alpha bungarotoxin; TTX, tetrodotoxin; PBS, phosphate buffered saline; NMJs, neuromuscular junctions; DFP, diisopropylfluorophosphate; PFA, paraformaldehyde; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; EPSPs, end plate postsynaptic potentials.1The abbreviations used are: AChE, acetylcholinesterase; AChR, acetylcholine receptor; ColQ, collagen Q; α-BTX, alpha bungarotoxin; TTX, tetrodotoxin; PBS, phosphate buffered saline; NMJs, neuromuscular junctions; DFP, diisopropylfluorophosphate; PFA, paraformaldehyde; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; EPSPs, end plate postsynaptic potentials. plays a critical role in the control of acetylcholine hydrolysis during synaptic transmission (1Katz B. Miledi R. J. Physiol. (Lond.). 1973; 231: 549-574Crossref Scopus (381) Google Scholar, 2Soreq H. Seidman S. Nat. Rev. Neurosci. 2001; 2: 294-302Crossref PubMed Scopus (1021) Google Scholar). The efficacy by which AChE controls neurotransmitter lifetime in the synaptic cleft depends not only on its enzymatic activity but also on its density and location relative to acetylcholine receptors (AChRs). Although progress has been made in elucidating the cellular and molecular events regulating AChR dynamics at the postsynaptic membrane (3Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1209) Google Scholar, 4Sanes J.R. Lichtman J.W. Nat. Rev. Neurosci. 2001; 2: 791-805Crossref PubMed Scopus (793) Google Scholar), our knowledge concerning the cellular basis of AChE dynamics is relatively limited. In particular it is unclear how this molecule is inserted and maintained at the synapse in vivo. At the neuromuscular junction, AChE is organized in a tetramer by collagen Q (ColQ) and is tethered in the extracellular matrix via ColQ and a complex of associated proteins, including perlecan, dystroglycan, and a muscle-specific tyrosine kinase (5Deprez P. Inestrosa N.C. Krejci E. J. Biol. Chem. 2003; 278: 23233-23242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 6Rotundo R.L. Rossi S.G. Anglister L. J. Cell Biol. 1997; 136: 367-374Crossref PubMed Scopus (36) Google Scholar, 7Cartaud A. Strochlic L. Guerra M. Blanchard B. Lambergeon M. Krejci E. Cartaud J. Legay C. J. Cell Biol. 2004; 165: 505-515Crossref PubMed Scopus (140) Google Scholar). Mutation or absence of ColQ or perlecan severely reduces the clustering of AChEs at neuromuscular junctions (NMJs) (8Ohno K. Brengman J. Tsujino A. Engel A.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9654-9659Crossref PubMed Scopus (224) Google Scholar, 9Feng G. Krejci E. Molgo J. Cunningham J.M. Massoulié J. Sanes J.R. J. Cell Biol. 1999; 144: 1349-1360Crossref PubMed Scopus (147) Google Scholar, 10Arikawa-Hirasawa E. Rossi S.G. Rotundo R.L. Yamada Y. Nat. Neurosci. 2002; 5: 119-123Crossref PubMed Scopus (142) Google Scholar). The availability of fasciculin2, a snake toxin purified from the green mamba (Dendroaspis angusticeps), and the presence of the large number of AChEs at the neuromuscular junction have enabled us to study directly the dynamics of AChE. Fasciculin2 belongs to the three-finger toxin family along with the well known acetylcholine receptor blocker, α-bungarotoxin (11Karlsson E. Mbugua P.M. Rodriguez-Ithurralde D. J. Physiol. (Paris). 1984; 79: 232-240PubMed Google Scholar), and has a high affinity for AChE (12Harel M. Kleywegt G.J. Ravelli R.B. Silman I. Sussman J.L. Structure (Lond.). 1995; 3: 1355-1366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 13Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 14Marchot P. Bourne Y. Prowse C.N. Bougis P.E. Taylor P. Toxicon. 1998; 36: 1613-1622Crossref PubMed Scopus (19) Google Scholar). When fasciculin2 binds AChE, 94% of enzyme activity is inhibited, allowing only residual detectable AChE activity (15Eastman J. Wilson E.J. Cervenansky C. Rosenberry T.L. J. Biol. Chem. 1995; 270: 19694-19701Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 16Rosenberry T.L. Rabl C.R. Neumann E. Biochemistry. 1996; 35: 685-690Crossref PubMed Scopus (30) Google Scholar). Previously, fasciculin2 has been used to characterize AChEs biochemically (11Karlsson E. Mbugua P.M. Rodriguez-Ithurralde D. J. Physiol. (Paris). 1984; 79: 232-240PubMed Google Scholar) as a probe to quantify AChE density in the mouse sternomastoid muscle (17Anglister L. Eichler J. Szabo M. Haesaert B. Salpeter M.M. J. Neurosci. Methods. 1998; 81: 63-71Crossref PubMed Scopus (26) Google Scholar), and fluorescently labeled fasciculin2 has been used to label AChEs on cultured cells, muscle cross-sections, and whole mounted muscle (18Peng H.B. Xie H. Rossi S.G. Rotundo R.L. J. Cell Biol. 1999; 145: 911-921Crossref PubMed Scopus (183) Google Scholar). In the present work, by saturating all pre-existing AChEs with unlabeled fasciculin2, we were able to monitor the subsequent insertion of new AChEs with fluorescently labeled fasciculin2. We found that AChEs were rapidly inserted into NMJs and that postsynaptic activity is necessary to enable normal AChE insertion through a mechanism mediated by intracellular calcium. Unless stated otherwise, compounds used in this study were obtained from Sigma. Unlabeled fasciculin2 (Latoxan, Valence France) and Alexa 594 fasciculin2 (conjugated by Molecular Probes Eugene, OR) were used to label AChE. Unlabeled and Alexa 488-conjugated bungarotoxin were obtained from Molecular Probes (Eugene, OR). To chelate cytosolic calcium, we used BAPTA-AM (Molecular Probes, Eugene, OR), a membrane-permeant substance that generates the high affinity calcium chelator BAPTA by the hydrolysis of ester bonds. As a control for any nonspecific effects of the hydrolysis products, we used Mag-Fura2-AM (Molecular Probes, Eugene, OR), which has a much lower affinity for calcium but generates the same by-products. In Vivo Imaging of Neuromuscular Junctions—Non-Swiss Albino adult female mice (6–10 weeks old, 25–30 g) were obtained from Harlan Sprague-Dawley. The mice were anesthetized with an intraperitoneal injection of a mixture of ketamine and xylazine (17.38 mg/ml). Sternomastoid muscle exposure and neuromuscular junction imaging were done as described in detail previously (19van Mier P. Lichtman J.W. J. Neurosci. 1994; 14: 5672-5686Crossref PubMed Google Scholar, 20Lichtman J.W. Magrassi L. Purves D. J. Neurosci. 1987; 7: 1215-1222Crossref PubMed Google Scholar, 21Akaaboune M. Culican S.M. Turney S.G. Lichtman J.W. Science. 1999; 286: 503-507Crossref PubMed Scopus (189) Google Scholar). Briefly, the anesthetized mouse was placed on its back on the stage of a customized epifluorescence microscope, and neuromuscular junctions were viewed under a coverslip with a water immersion objective (×20 UAPO 0.7 NA Olympus BW51, Optical Analysis Corp., Nashua, NH) and digital CCD camera (Retiga EXi, Burnaby, British Columbia, Canada). All animal usage followed methods approved by the University of Michigan Committee on the Use and Care of Animals. Mice were intubated and ventilated for the duration of the imaging sessions. For experiments in which the junction was to be re-imaged within 8 h, the animal was continuously ventilated and maintained under anesthesia by intraperitoneal doses of ketamine and xylazine every 2 h. To minimize evaporation, the muscle was bathed with lactated Ringer's containing whatever drugs were appropriate to the experiment, and a coverslip was placed over the exposed muscle. A fresh dose of drug solution was added every 2 h. For multiple time points beyond 8 h, the mouse was sutured and allowed to fully recover before the next imaging session. In some experiments, the sternomastoid muscle was stimulated by a Grass SD5 stimulator connected to two platinum wires at either side of the muscle. The stimulus pulses (3-ms bipolar pulses of 6–9 V at 10 Hz for a 1-s duration every 2 s) elicited maximal twitching and therefore action potentials in all muscle fibers. Quantitative Fluorescence Imaging—The fluorescence intensity of labeled AChE at neuromuscular junctions was assayed by using a quantitative fluorescence imaging technique, as described by Turney and colleagues (22Turney S.G. Culican S.M. Lichtman J.W. J. Neurosci. Methods. 1996; 64: 199-208Crossref PubMed Scopus (39) Google Scholar) with minor modification. This technique incorporates compensation for image variation that may be caused by spatial and temporal changes in the light source and camera between imaging sessions by calibrating the images with a nonfading reference standard. Image analysis was performed by using either a procedure written for IPLAB (Scanalytic, VI) or Matlab (The Mathworks, Natick, MA). Background fluorescence was approximated by selecting a boundary region around the junction and subtracting it from the original image, and the mean of the total fluorescence intensity (which corresponds to density) was measured. After saturating all pre-existing AChEs with unlabeled fasciculin2 and re-saturating newly inserted AChEs with fluorescent fasciculin2 at a later time, the mean of the total fluorescence of newly labeled AChE was expressed as the percentage of the mean of fluorescence of AChEs saturated with only Alexa 594 fasciculin2 at time 0. When average intensity is presented, it is ± S.D. The Rate of Fasciculin2 Unbinding from Synapses Is Extremely Slow—The interpretation of the experiments described in this study depends on the rate of unbinding of unlabeled fasciculin2 from AChE. Based on two lines of evidence, we conclude that the rate of unbinding of unlabeled fasciculin2 is slow enough to be considered negligible over the 7-day time span we made the measurements reported in the rest of this study. The first group of experiments was conducted on muscles dissected free of the animal, fixed, and then washed and maintained in 0.5 m glycine PBS (Fig. 1). The advantage of studying fixed tissue is that new synthesis and insertion of AChE are eliminated, so determining the rate of unbinding of fasciculin2 under these conditions is simple. We first demonstrated that neither the ability of Alexa 594-labeled fasciculin2 to interact with AChE nor the ability of Alexa 488-labeled α-bungarotoxin (α-BTX) to interact with AChR was inhibited by four different fixation conditions. In Fig. 1, muscles fixed with 2% paraformaldehyde (PFA) in PBS are shown, but similar results were obtained with 4% PFA, 8% PFA, or 2% PFA and 2% glutaraldehyde in PBS (although in the latter case autofluorescence was extremely high). Muscles fixed, washed, and labeled immediately with Alexa 594-conjugated fasciculin2 (Fig. 1, A and B) and muscles fixed, washed, and then maintained in PBS for as long as 2 months before being exposed to Alexa 594-conjugated fasciculin2 (Fig. 1, C and D) labeled with approximately equal intensity, indicating that fixation does not affect the toxin-binding sites of AChE or AChR. The AChE staining was specific, because it could be prevented by adding saturating levels of unlabeled fasciculin2 or BW284C51 (an inhibitor of AChE that competes with fasciculin2) just prior to adding Alexa 594-conjugated fasciculin2. Binding of Alexa 488 α-BTX was not affected by fasciculin2 (Fig. 1, E–H). The key experiment was to add unlabeled fasciculin2 shortly after washing off the fixative and then probing with Alexa 594-labeled fasciculin2 at a later time. All sites from which the unlabeled fasciculin2 had unbound and washed away should be stained with Alexa 594 fasciculin2. However, even when 2 weeks were allowed for unbinding, there was little or no detectable labeling (Fig. 1J). The dissociation rate we observed for fasciculin2 from AChE at synapses in fixed tissue is far slower than the published unbinding rate of fasciculin2 from solubilized AChE in a test tube, which yields half-lives in the range of a few hours (12Harel M. Kleywegt G.J. Ravelli R.B. Silman I. Sussman J.L. Structure (Lond.). 1995; 3: 1355-1366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 13Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 14Marchot P. Bourne Y. Prowse C.N. Bougis P.E. Taylor P. Toxicon. 1998; 36: 1613-1622Crossref PubMed Scopus (19) Google Scholar, 23Marchot P. Khelif A. Ji Y.H. Mansuelle P. Bougis P.E. J. Biol. Chem. 1993; 268: 12458-12467Abstract Full Text PDF PubMed Google Scholar, 24Radic Z. Duran R. Vellom D.C. Li Y. Cervenansky C. Taylor P. J. Biol. Chem. 1994; 269: 11233-11239Abstract Full Text PDF PubMed Google Scholar, 25Radic Z. Quinn D.M. Vellom D.C. Camp S. Taylor P. J. Biol. Chem. 1995; 270: 20391-20399Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 26Radic Z. Kirchhoff P.D. Quinn D.M. McCammon J.A. Taylor P. J. Biol. Chem. 1997; 272: 23265-23277Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). A potential explanation for the slow apparent rate of unbinding from intact synapses would be that the actual rate of unbinding is much more rapid, but that most of the time the fasciculin2 is rebound rather than diffusing away. To test this possibility, muscles were fixed with 2% PFA, washed, saturated with unlabeled fasciculin2, washed again, and then incubated continuously with a high concentration of Alexa 594 fasciculin2. Under these conditions, it is expected that any fasciculin2 that unbinds will be replaced by Alexa 594 fasciculin2, so the rate of appearance of fluorescence will provide an estimate of the rate of dissociation of unlabeled toxin uncontaminated by rebinding. When we imaged synapses that were continuously incubated in Alexa 594 fasciculin2 for 4 days, we saw little or no evidence for AChEs staining with fluorescent fasciculin2 (Fig. 1L). The second group of experiments tested whether the extremely slow rate of loss of fasciculin2 was a peculiarity of fixed muscle. We placed living mouse diaphragm muscles into organ culture. Synapses were saturated with unlabeled fasciculin2, washed, and then continuously exposed for 24 h to Alexa 488-labeled fasciculin2 in the presence or absence of the protein synthesis inhibitor cycloheximide (Fig. 2). The rationale for these experiments is that in the absence of cycloheximide, the gain of fluorescence over 24 h might result from three processes as follows: 1) the loss of unlabeled fasciculin2 from pre-existing AChEs followed by rebinding of labeled fasciculin2; 2) the transfer of already synthesized AChEs that were in an intracellular compartment to the cell surface; and 3) the accumulation of newly synthesized AChEs on the surface. Cycloheximide will eliminate or dramatically affect this third possible pathway. In the absence of cycloheximide (Fig. 2, A–C), there was intensive synaptic labeling with Alexa 488-labeled fasciculin2. On average, the intensity was 18% that of control labeled with only Alexa 488-fasciculin2 (that had not been exposed to unlabeled fasciculin2). After 24 h in the presence of cycloheximide, the Alexa 488 fluorescence was barely detectable (Fig. 2, D–I). The average intensity of the Alexa 488 fasciculin2-labeled synapses from cycloheximide-treated muscles was 1–3% of noncycloheximide-treated muscle. If this entire signal was because of fasciculin2 dissociation that followed an exponential time course, this would correspond to a half-life of about 35 days. Because the transfer of pre-existing AChEs to the surface also contributes to this signal, the actual half-life for dissociation is even longer than this. Thus, these experiments were sufficient to conclude that the loss of unlabeled fasciculin2 from AChE in living muscle must be very slow, just as was the case for fixed muscle. AChE Insertion at the Neuromuscular Junction in Living Mice following a Single Saturating Dose of Fasciculin2—To determine the number of new AChEs inserted into the neuromuscular junctions of living animals over time, the sternomastoid muscle of six mice at each data point was bathed with unlabeled fasciculin2 (7 μg/ml, 2.5 h) to saturate all pre-existing AChEs. We confirmed that all AChEs were saturated by adding a fluorescently conjugated Alexa 594 fasciculin2 (7 μg/ml, 10 min) and demonstrating an absence of red fluorescence. Because the rate of unbinding of unlabeled fasciculin2 is undetectably slow (see above), any red fluorescence detected after re-labeling the muscle with Alexa 594 fasciculin2 at a later time must come from the binding of fasciculin2 to newly inserted AChE. The new AChE will include both newly synthesized molecules and AChE that was already present but not on the surface. The number of AChEs inserted after initial saturation of the NMJ with unlabeled fasciculin2 was expressed as a percentage of the fluorescence present when control neuromuscular junctions were saturated with Alexa 594 fasciculin2 and immediately imaged (Fig. 3, A and B). After saturating all pre-existing AChEs with unlabeled fasciculin2 and re-saturating newly inserted AChEs with Alexa 594 fasciculin2 4 h later, we found that the recovery of fluorescence was ∼6 ± 3% (n = 50) of the total fluorescence of AChEs saturated with only Alexa 594 fasciculin2 (Fig. 3, A and B). At 8 and 24 h and 3 and 7 days after saturating all pre-existing AChEs with unlabeled fasciculin2, the recovery of fluorescence intensity was, respectively, 11 ± 3% (n = 40), 20 ± 3% (n = 15), 35 ± 5% (n = 30), and 65 ± 9% (n = 25) of total fluorescence at synapses saturated only with Alexa 594 fasciculin2 (Fig. 3, A and B). This AChE recovery corresponds to an initial rate of about 2% per h (t½ ∼46 h), which slowed by 7 days to 0.7% per h (t½ ∼7 days). Our results showing that AChE insertion is initially rapid differ dramatically from previous reports claiming that the insertion of AChE does not occur until 3–7 days after initial blockade with DFP, an organophosphate esterase inhibitor, and that the rate of insertion is very slow at all times (27Rogers A.W. Darzynkiewicz Z. Salpeter M.M. Ostrowski K. Barnard E.A. J. Cell Biol. 1969; 41: 665-685Crossref PubMed Scopus (29) Google Scholar) (see “Discussion”). AChE Insertion into the Neuromuscular Junction Depends on Muscle Postsynaptic Activity—Because many processes in skeletal muscle are regulated by synaptic activity, we carried out three types of experiments to test the role of activity in regulating the rate of appearance of new AChE. All three approaches used toxins that decrease muscle activity by different mechanisms. α-BTX (10 μg/ml) blocks nAChRs, and so a saturating dose directly eliminates evoked and miniature end plate postsynaptic potentials (EPSPs) and indirectly eliminates action potentials in the muscle. TTX (1 μg/ml) blocks voltage-gated sodium channels on both axons and on muscle fibers, and so also indirectly blocks evoked EPSPs, while leaving miniature EPSPs intact. μ-Conotoxin GIIIB (2 μm) selectively blocks voltage-gated sodium channels on muscle fibers, while sparing those on axons, leaving neuronal action potentials and both evoked and miniature EPSPs unchanged while blocking muscle action potentials (28Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar). The experimental paradigm used to test the effect of exogenous agents on AChE insertion was to apply a saturating dose of unlabeled fasciculin2 (7 μg/ml, 2.5 h) to the sternomastoid muscle followed by a second dose of Alexa 594 fasciculin2 (7 μg/ml, 10–20 min), to be sure that all synapses were saturated with unlabeled fasciculin2, and then to continuously bathe the muscle with a toxin for 8 h (with reapplication every 2 h to make sure that the agent was present continuously). At the end of this period, Alexa 594 fasciculin2 was applied at a saturating dose to label all AChEs that had been inserted over that time. These treatments required that animals be continuously ventilated and could not be carried beyond 8 h because of mortality. The fluorescence of synapses in toxin-treated animals was approximately half the fluorescence of synapses in control animals labeled similarly but not treated with any toxin (α-BTX 46 ± 7% (n = 40); TTX 50 ± 8% (n = 25); and μ-conotoxin GIIIB 46 ± 8% (n = 35); Fig. 4, A–F). Thus new AChE can appear in the absence of spike activity in the muscle at a basal rate, but the rate of AChE appearance was doubled when muscle action potentials were not blocked. Neither presynaptic spikes nor synaptic potentials were able to increase the appearance of new AChE above the basal level if muscle action potentials were absent. Muscle Postsynaptic Activity Restores AChE Insertion—As a final test of the role of muscle spike activity on AChE insertion, the sternomastoid muscle was first bathed with unlabeled fasciculin2 to saturate all pre-existing AChEs, and postsynaptic muscle activity was eliminated by blocking synaptic transmission with a high dose of α-bungarotoxin (10 μg/ml), and then action potentials were directly elicited by placing stimulating electrodes at either end of the muscle (3-ms bipolar pulses of 6–9 V at 10 Hz for 1-s duration every 2 s for the entire 8-h period). During the duration of this experiment, the mouse was intubated and ventilated to prevent asphyxia. We found that the level of expression of AChE in these muscles (112 ± 16%, n = 40) was approximately double the level found in muscles treated with α-BTX alone and was not significantly different from untreated control muscles (Fig. 5, A and B). This result indicates that muscle action potentials are necessary and sufficient to maintain normal levels of AChE insertion. Intracellular Calcium Levels Are Critical for AChE Insertion—Muscle contraction is the consequence of an elevation of intracellular calcium. It therefore seemed possible that the molecular mechanism by which muscle activity modulates AChE insertion also requires an elevation of intracellular calcium. To test this hypothesis, four different types of experiments were carried out. One way for calcium to become elevated is by entering the cell through voltage-activated channels in the plasma membrane. In innervated adult mouse skeletal muscle, only one type of voltage-gated calcium channel (the L-type channel that is slowly activated and inactivated by depolarization and can be blocked by dihydropyridines and verapamil) has been described (29Rotzler S. Schramek H. Brenner H.R. Nature. 1991; 349: 337-339Crossref PubMed Scopus (56) Google Scholar, 30McCleskey E.W. J. Physiol. (Lond.). 1985; 361: 231-249Crossref Scopus (44) Google Scholar, 31Beam K.G. Knudson C.M. J. Gen. Physiol. 1988; 91: 781-798Crossref PubMed Scopus (128) Google Scholar, 32Cognard C. Lazdunski M. Romey G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 517-521Crossref PubMed Scopus (125) Google Scholar). To determine the potential role of Ca2+ flux through this channel on AChE insertion rates, the sternomastoid muscle was incubated with unlabeled fasciculin2 to saturate all pre-existing AChEs and then electrically stimulated in the presence of a high dose of unlabeled bungarotoxin (to block calcium entry through AChRs) and verapamil (50 μm) (to block calcium entry through voltage-gated L-type Ca2+ channels) for the duration of the experiment. Eight hours later, newly inserted AChEs were labeled with Alexa 594 fasciculin2, and their fluorescence intensity was measured. We found that the fluorescence of newly inserted AChE was only 45 ± 8% (n = 23) of the total fluorescence of control muscle synapses labeled after 8 h with Alexa 594 fasciculin2 (Fig. 6, A and C). However, in unstimulated muscle this drug had little effect on AChE insertion. These results suggest that calcium entry through voltage-dependent calcium channels is involved in AChE insertion. A second mechanism for elevating intracellular free calcium is Ca2+ release from the sarcoplasmic reticulum. To investigate whether the release of intracellular calcium from the sarcoplasmic reticulum in the absence of muscle activity can modulate AChE insertion, muscle postsynaptic activity was chronically blocked with unlabeled α-BTX in the presence of ryanodine, a plant alkaloid known to release calcium from the sarcoplasmic reticulum (33Fairhurst A.S. Am. J. Physiol. 1974; 227: 1124-1131Crossref PubMed Scopus (27) Google Scholar, 34Fairhurst A.S. Biochem. Pharmacol. 1973; 22: 2815-2827Crossref PubMed Scopus (9) Google Scholar, 35Jenden D.J. Fairhurst A.S. Pharmacol. Rev. 1969; 21: 1-25PubMed Google Scholar). Treatment with 0.3 μm ryanodine, a concentration known to tonically elevate intracellular free calcium (36Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Abstract Full Text PDF PubMed Google Scholar), resulted in AChE insertion levels comparable with those of control muscles (115 ± 18%, n = 38) and double those in muscles treated with α-BTX alone, indicating that directly releasing calcium from intracellular stores can bypass the need for spike activity to elevate AChE insertion (Fig. 6, B and C). Verapamil was unable to inhibit the effect of 0.3 μm ryanodine, arguing that the calcium flux through L-type channels is not required to replenish the intracellular calcium stores in the sarcoplasmic reticulum. In contrast, treatment with 100 μm ryanodine, which transiently elevates intracellular free calcium but then leaves the stores depleted for many hours (36Meissner G. J. Biol. Chem. 1986; 261: 6300-6306Abstract Full Text PDF PubMed Google Scholar), resulted in only basal levels of AChE insertion or Ca2+ concentration (data not shown). A third test of the involvement of intracellular free calcium in AChE insertion was to bypass all normal cellular pathways and to elevate intracellular free calcium directly by adding the Ca2+ ionophore A23187 (3 μm). Treatment of α-BTX-blocked muscles with A23187 elicited an increase in the expression of AChE to about double the base-line value (100 ± 7%, n = 25) (Fig. 6, D and F), as would be expected if a rise in intracellular free calcium plays a role in AChE insertion. Finally, we examined the effect of adding an exogenous calcium buffer into the muscle cells, with the expectation that this might attenuate the spike-induced increase in intracellular free calcium. Chronic exposure to BAPTA-AM (500 μm) for 8 h decreased the level of AChE insertion to 17 ± 5% (n = 20) (Fig. 6, E and F) of control fluorescence, significantly below the basal level of about 50% seen in all other treatments that blocked activity. This effect was specific, as the treatment with Mag-Fura2-AM, which would produce the same waste product as BAPTA-AM but has a much lower affinity for calcium than BAPTA and thus should be incapable of chelating physiological levels of calcium, had no effect on AChE insertion. A" @default.
• W2170918734 created "2016-06-24" @default.
• W2170918734 creator A5001675109 @default.
• W2170918734 creator A5017035439 @default.
• W2170918734 creator A5029096406 @default.
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• W2170918734 date "2005-09-01" @default.
• W2170918734 modified "2023-10-01" @default.
• W2170918734 title "In Vivo Regulation of Acetylcholinesterase Insertion at the Neuromuscular Junction" @default.
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