In vivo recovery of muscle contraction after alpha-bungarotoxin binding.

Fertuck, Helen C.; Woodward, William R.; Salpeter, Miriam M.

doi:10.1083/jcb.66.1.209

Cited by 35 publications

(18 citation statements)

References 10 publications

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“…This can be seen from the ratio of sites per square micrometer at the thickened and nonthickened membranes (Tables I and II) and from the result given in the text that there is a .high positive label at the top of the thickened pjm (source compartment 2) at new zero time. (A similar conclusion was reached in an earlier study [17].) In that sense, the adult innervated junction appears to differ from the developing ectopic junction (42).…”

Section: Insertion Of New Receptorssupporting

confidence: 68%

Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction.

Salpeter¹,

Harris²

1983

The Journal of Cell Biology

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ABSIRACI The distribution and turnover rate of acetylcholine receptors labeled with t2Sl-~x-bungarotoxin were examined in innervated mouse sternomastoid muscle by electron microscope autoradiography using the "mask" analysis procedure. We compared the total population of receptors with receptors newly inserted at the junction 2 d after inactivation with nonradioactive cz-bungarotoxin, both at the top {thickened) region of the postjunctional folds (pjm) and the nonthickened bottom folds. We found that the receptor site density was -10 times greater on the thickened pjm than on the nonthickened bottom folds for both total and newly inserted receptors. This ratio does not change significantly during a 6-d period after labeling the new receptors. Furthermore, calculated values for turnover time of receptors show that both total and newly inserted receptors at both regions of the junctional folds have half-lives for degradation within the range given in the literature for slow junctional receptors. These data exclude a simple migration model whereby receptors are preferentially inserted in the nonthickened region of the junctional folds and then migrate into the thickened membrane at a rate equal to the turnover-rate of the receptors.The postsynaptic folds of the vertebrate neuromuscular junction are heterogeneous in terms of both receptor distribution and morphology. The top ~2,000 A of postjunctional membrane (pjm) has a distinct electron density, a cytoplasmic filamentous substructure (4) and is rich in intramembrane particles (13,26,28). This thickened region has a high density of receptors (~10,000-20,000 sites/#m 2) (2,15,16, 19,23,27). At the bottom of the folds, the membrane is morphologically indistinguishable from that at extrajunctional regions, and the receptor site density is considerably reduced (~ 1,000 sites//.tm 2, or ~5-10% the site density at the top of the folds). This site density is equivalent to that seen in the perijunctional region, 0.5-1 #m immediately surrounding each axonal end bouton (16).The reason for the heterogeneity in the organization of the pjm has not yet been established. Much is known about the properties of extrajunctional and junctional receptors both during development and after denervation (for review see references 10, 14). However, nothing is known about the properties of the acetylcholine receptor (AChR) at the bottom, nonthickened region of the pjm. In the present study, we asked two questions: (a) Do the AChRs in the nonthickened bottom folds resemble extrajunctional or embryonic receptors in having a fast turnover rate, and (b) do the nonthickened bottom folds provide a region for the insertion of AChRs that subsequently migrate to the thickened pjm7 We used innervated mouse sternomastoid muscle to examine both the localization and turnover rate of junctional receptors at the top thickened pjm and at the bottom nonthickened membrane. We did this for the total population of receptors as well as for receptors newly inserted at the junction during a 2-d period. We found t...

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Section: Insertion Of New Receptorssupporting

confidence: 68%

Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction.

Salpeter¹,

Harris²

1983

The Journal of Cell Biology

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“…Five mice were used in this study. In two mice the sternomastoid muscle of the anesthetized animals was exposed by an incision in the neck, and ~251-ct-BTX (0.8-1.6 • 10 -e M) was applied topically until a complete block of neurally evoked tetanic muscle contraction was obtained (see reference 23). To determine when this physiological endpoint was reached, the uncut nerve was intermittently stimulated once every 15 min via a suction electrode.…”

Section: Preparation Of Tissuementioning

confidence: 99%

Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions

Fertuck¹,

Salpeter²

1976

The Journal of Cell Biology

394

223

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The distribution and quantitation of l~q-a-bungarotoxin (a-BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetanic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, ct-BTX was bound to ~90% of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of a-BTX relative to this membrane was determined to be 46,000 + 27% sites/~m ~.By testing various hypothetical models the distribution of developed grains was found to be consistent with the hypothesis that the main acetylcholine-receptive surface coincides with the electron-dense, thickened portion of the junctional fold membrane situated at the juxtaneuronal region of the folds and dipping ,-,2,200 ,~ down the folds. The concentration of a-BTX binding sites was then calculated to be -,-30,500 • 27% sites/~m ~ of this postsynaptic dense membrane.There was a sharp gradient in ct-BTX binding extrajunctionally, with the concentration decreasing to ,-,4% of the subsynaptic value within 1 tzm from the edge of an axonal terminal (bouton) to < 1% within 3 ~m and to <0.2% beyond 7 #m from that terminal. Below 4,000 A (i.e. half-way from the top of the junctional folds) the concentration of a-BTX was also about 3% of the peak subsynaptic value. The binding density at the bottom of the junctional folds is thus comparable to extrajunctional sarcolemma at equal distance from a nerve terminal. The molecular organization at the neuromuscular junction relative to its function is discussed.In neuromuscular junctions of vertebrate twitch muscle, the motor axon bifurcates terminally and ends in numerous elongated boutons within gutters formed by the invaginations of the muscle surface.Characteristic of the subneural muscle membrane are deep infoldings (junctional folds) which extend into the muscle for about 0.5-1.0 ~m. Junctional folds are absent in the interneuronal regions of the 144

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“…The toxin obtained from Bungarus multicinctus, x-bungarotoxin (a-BTX) (Chang & Lee, 1963; Lee, Chang, Kau & Shing-Hui Luh, 1972), as well as Cobra venom (Chang & Lee, 1963;Lee & Chang, 1966; Meldrum, 1965;Lester, 1970;Eaken, Manvis & Thesleff, 1971;Earl & Excell, 1972) have been shown to produce a post-junctional blockade at skeletal neuromuscular junctions which resembles that of curare. These toxins are known to bind specifically to nicotinic receptors for ACh in electric tissue of fish (Changeux, Kasai & Lee, 1970;Miledi, Molinoff & Potter, 1971;Lester, 1970) and the end-plate regions of vertebrate skeletal muscle (Chang & Lee, 1963;Bernard, Wieckowski & Chiu, 1971;Berg, Kelly, Sangent, Williamson & Hall, 1972; Lee, 1972;Berg & Hall, 1975; Lee, Tseng & Chiu, 1967;Fertuck, Woodward & Salpeter, 1975;Fambrough & Hartzell, 1972). This highly specific binding has permitted the toxins, particularly a-bungarotoxin, to be used to localize nicotinic receptors in 18 skeletal muscle.…”

Section: Introductionmentioning

confidence: 99%

“…This highly specific binding has permitted the toxins, particularly a-bungarotoxin, to be used to localize nicotinic receptors in 18 skeletal muscle. Radiolabelled toxin and radioautography (Lee et al 1967;Fertuck & Salpeter, 1974;Fertuck et al 1975;Fambrough & Hartzell, 1972) or fluorescein-labelled toxin (Anderson & Cohen, 1974) have been used for this purpose.…”

Section: Introductionmentioning

confidence: 99%

Discrimination between nicotinic receptors in vertebrate ganglia and skeletal muscle by alpha‐bungarotoxin and cobra venoms.

Bursztajn¹,

Gershon²

1977

The Journal of Physiology

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In vivo recovery of muscle contraction after alpha-bungarotoxin binding.

Cited by 35 publications

References 10 publications

Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction.

Distribution and turnover rate of acetylcholine receptors throughout the junction folds at a vertebrate neuromuscular junction.

Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions

Discrimination between nicotinic receptors in vertebrate ganglia and skeletal muscle by alpha‐bungarotoxin and cobra venoms.

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