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The presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists in skinned rat skeletal muscle fibres.

Duke AM, Steele DS - Cell Calcium (2008)

Bottom Line: When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05).However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished.These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

View Article: PubMed Central - PubMed

Affiliation: Institute of Membrane and Systems Biology, University of Leeds, Woodhouse Lane, Leeds LS29JT, United Kingdom.

ABSTRACT
Single mechanically skinned extensor digitorum Longus (EDL) rat fibres were used as a model to study the influence of functional t-tubules on the properties of RyR1 in adult skeletal muscle. Fibres were superfused with solutions approximating to the intracellular milieu. Following skinning, the t-tubules re-seal and repolarise, allowing the sarcoplasmic reticulum (SR) Ca2+ release to be activated by field stimulation. However, in the present study, some fibres exhibited localised regions where depolarisation-induced SR Ca2+ release was absent, due to failure of the t-tubules to re-seal. When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05). This difference in RyR1 sensitivity was unaffected by sustained depolarisation of the t-tubule network. However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished. These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

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Effects of t-tubule permeabilization or Ca2+ depletion on SR Ca2+ release. (A) A mechanically skinned fibre was field-stimulated to identify a region exhibiting SR Ca2+ release failure. Selected x–y images are shown under control conditions and during the peak of responses to, tetanic field stimulation, 5 mM caffeine or 20 mM caffeine/low Mg2+ (upper panel). The same preparation was then exposed to 50 μg/ml saponin, which resulted in a localised rise in [Ca2+] due to permeabilization and depolarisation of the re-sealed t-tubules. After saponin treatment, neither field stimulation nor substitution of K-HDTA with Na-HDTA elicited a response, consistent with irreversible disruption of the t-tubule network (middle panel). After saponin treatment, both submaximal responses to 2 or 5 mM caffeine and the maximal response to 20 mM caffeine/low Mg2+ were spatially uniform (lower panel). Similar results were obtained in four other cells. (B) A fibre was mechanically skinned in the presence of 5 mM EGTA in order to reduce the [Ca2+] within the t-tubule network to nanomolar levels. Following skinning, field-stimulation was used to identify localised regions of SR Ca2+ release failure. On addition of 5 mM caffeine, the resulting Ca2+ release was uniform. Similar results were obtained in four other cells. (C) A fibre was exposed to Tyrode's solution, containing 10 mM total Mg2+ (8.7 mM free) and 5 mM EGTA before skinning. Field stimulation was again used to identify localised Ca2+ release failure. In the presence of 8.7 mM t-tubule Mg2+, localised regions exhibiting Ca2+ release failure were apparent, and regions exhibiting SR Ca2+ release in response to field stimulation were more sensitive to caffeine. Similar results were obtained in four other cells.
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fig5: Effects of t-tubule permeabilization or Ca2+ depletion on SR Ca2+ release. (A) A mechanically skinned fibre was field-stimulated to identify a region exhibiting SR Ca2+ release failure. Selected x–y images are shown under control conditions and during the peak of responses to, tetanic field stimulation, 5 mM caffeine or 20 mM caffeine/low Mg2+ (upper panel). The same preparation was then exposed to 50 μg/ml saponin, which resulted in a localised rise in [Ca2+] due to permeabilization and depolarisation of the re-sealed t-tubules. After saponin treatment, neither field stimulation nor substitution of K-HDTA with Na-HDTA elicited a response, consistent with irreversible disruption of the t-tubule network (middle panel). After saponin treatment, both submaximal responses to 2 or 5 mM caffeine and the maximal response to 20 mM caffeine/low Mg2+ were spatially uniform (lower panel). Similar results were obtained in four other cells. (B) A fibre was mechanically skinned in the presence of 5 mM EGTA in order to reduce the [Ca2+] within the t-tubule network to nanomolar levels. Following skinning, field-stimulation was used to identify localised regions of SR Ca2+ release failure. On addition of 5 mM caffeine, the resulting Ca2+ release was uniform. Similar results were obtained in four other cells. (C) A fibre was exposed to Tyrode's solution, containing 10 mM total Mg2+ (8.7 mM free) and 5 mM EGTA before skinning. Field stimulation was again used to identify localised Ca2+ release failure. In the presence of 8.7 mM t-tubule Mg2+, localised regions exhibiting Ca2+ release failure were apparent, and regions exhibiting SR Ca2+ release in response to field stimulation were more sensitive to caffeine. Similar results were obtained in four other cells.

Mentions: In the experiment shown in Fig. 5A, a skinned fibre was field stimulated, allowing a localised region lacking SR Ca2+ release to be identified. As in previous examples, addition of 5 mM caffeine induced Ca2+ release only in the region sensitive to field-stimulation, while 20 mM caffeine/low Mg2+ induced a uniform SR Ca2+ release throughout the preparation (upper panel). The preparation was then returned to a control solution before exposure to 50 μg/ml saponin for 10 min (middle panel). The initial exposure to saponin induced a detectable release of Ca2+ due to selective permeabilization (and depolarisation) of the re-sealed t-tubule network. Following removal of saponin, neither substitution of K-HDTA with Na-HDTA nor field stimulation induced a detectable rise in [Ca2+], consistent with permeabilization of the t-tubule network. However, after exposure to saponin, submaximal concentrations of caffeine (2 or 5 mM) induced a uniform release of Ca2+ throughout the fibre (lower panel). Exposure to 20 mM caffeine/low Mg2+ induced a maximal release of Ca2+, which was comparable to that obtained prior to saponin treatment, suggesting that saponin treatment did not affect the SR Ca2+ content or the distribution of Ca2+ within the SR network. Similar results were obtained in four other preparations.


The presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists in skinned rat skeletal muscle fibres.

Duke AM, Steele DS - Cell Calcium (2008)

Effects of t-tubule permeabilization or Ca2+ depletion on SR Ca2+ release. (A) A mechanically skinned fibre was field-stimulated to identify a region exhibiting SR Ca2+ release failure. Selected x–y images are shown under control conditions and during the peak of responses to, tetanic field stimulation, 5 mM caffeine or 20 mM caffeine/low Mg2+ (upper panel). The same preparation was then exposed to 50 μg/ml saponin, which resulted in a localised rise in [Ca2+] due to permeabilization and depolarisation of the re-sealed t-tubules. After saponin treatment, neither field stimulation nor substitution of K-HDTA with Na-HDTA elicited a response, consistent with irreversible disruption of the t-tubule network (middle panel). After saponin treatment, both submaximal responses to 2 or 5 mM caffeine and the maximal response to 20 mM caffeine/low Mg2+ were spatially uniform (lower panel). Similar results were obtained in four other cells. (B) A fibre was mechanically skinned in the presence of 5 mM EGTA in order to reduce the [Ca2+] within the t-tubule network to nanomolar levels. Following skinning, field-stimulation was used to identify localised regions of SR Ca2+ release failure. On addition of 5 mM caffeine, the resulting Ca2+ release was uniform. Similar results were obtained in four other cells. (C) A fibre was exposed to Tyrode's solution, containing 10 mM total Mg2+ (8.7 mM free) and 5 mM EGTA before skinning. Field stimulation was again used to identify localised Ca2+ release failure. In the presence of 8.7 mM t-tubule Mg2+, localised regions exhibiting Ca2+ release failure were apparent, and regions exhibiting SR Ca2+ release in response to field stimulation were more sensitive to caffeine. Similar results were obtained in four other cells.
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Related In: Results  -  Collection

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Show All Figures
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fig5: Effects of t-tubule permeabilization or Ca2+ depletion on SR Ca2+ release. (A) A mechanically skinned fibre was field-stimulated to identify a region exhibiting SR Ca2+ release failure. Selected x–y images are shown under control conditions and during the peak of responses to, tetanic field stimulation, 5 mM caffeine or 20 mM caffeine/low Mg2+ (upper panel). The same preparation was then exposed to 50 μg/ml saponin, which resulted in a localised rise in [Ca2+] due to permeabilization and depolarisation of the re-sealed t-tubules. After saponin treatment, neither field stimulation nor substitution of K-HDTA with Na-HDTA elicited a response, consistent with irreversible disruption of the t-tubule network (middle panel). After saponin treatment, both submaximal responses to 2 or 5 mM caffeine and the maximal response to 20 mM caffeine/low Mg2+ were spatially uniform (lower panel). Similar results were obtained in four other cells. (B) A fibre was mechanically skinned in the presence of 5 mM EGTA in order to reduce the [Ca2+] within the t-tubule network to nanomolar levels. Following skinning, field-stimulation was used to identify localised regions of SR Ca2+ release failure. On addition of 5 mM caffeine, the resulting Ca2+ release was uniform. Similar results were obtained in four other cells. (C) A fibre was exposed to Tyrode's solution, containing 10 mM total Mg2+ (8.7 mM free) and 5 mM EGTA before skinning. Field stimulation was again used to identify localised Ca2+ release failure. In the presence of 8.7 mM t-tubule Mg2+, localised regions exhibiting Ca2+ release failure were apparent, and regions exhibiting SR Ca2+ release in response to field stimulation were more sensitive to caffeine. Similar results were obtained in four other cells.
Mentions: In the experiment shown in Fig. 5A, a skinned fibre was field stimulated, allowing a localised region lacking SR Ca2+ release to be identified. As in previous examples, addition of 5 mM caffeine induced Ca2+ release only in the region sensitive to field-stimulation, while 20 mM caffeine/low Mg2+ induced a uniform SR Ca2+ release throughout the preparation (upper panel). The preparation was then returned to a control solution before exposure to 50 μg/ml saponin for 10 min (middle panel). The initial exposure to saponin induced a detectable release of Ca2+ due to selective permeabilization (and depolarisation) of the re-sealed t-tubule network. Following removal of saponin, neither substitution of K-HDTA with Na-HDTA nor field stimulation induced a detectable rise in [Ca2+], consistent with permeabilization of the t-tubule network. However, after exposure to saponin, submaximal concentrations of caffeine (2 or 5 mM) induced a uniform release of Ca2+ throughout the fibre (lower panel). Exposure to 20 mM caffeine/low Mg2+ induced a maximal release of Ca2+, which was comparable to that obtained prior to saponin treatment, suggesting that saponin treatment did not affect the SR Ca2+ content or the distribution of Ca2+ within the SR network. Similar results were obtained in four other preparations.

Bottom Line: When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05).However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished.These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

View Article: PubMed Central - PubMed

Affiliation: Institute of Membrane and Systems Biology, University of Leeds, Woodhouse Lane, Leeds LS29JT, United Kingdom.

ABSTRACT
Single mechanically skinned extensor digitorum Longus (EDL) rat fibres were used as a model to study the influence of functional t-tubules on the properties of RyR1 in adult skeletal muscle. Fibres were superfused with solutions approximating to the intracellular milieu. Following skinning, the t-tubules re-seal and repolarise, allowing the sarcoplasmic reticulum (SR) Ca2+ release to be activated by field stimulation. However, in the present study, some fibres exhibited localised regions where depolarisation-induced SR Ca2+ release was absent, due to failure of the t-tubules to re-seal. When these fibres were exposed to caffeine to directly activate RyR1, regions with re-sealed t-tubules exhibited greater sensitivity to submaximal (2-5 mM) levels of caffeine (n = 8), while the response to a supramaximal SR Ca2+ release stimulus was uniform (n = 8, p < 0.05). This difference in RyR1 sensitivity was unaffected by sustained depolarisation of the t-tubule network. However, after saponin permeabilization of the t-tubules or withdrawal of Ca2+ from the t-tubules before skinning, the difference in agonist sensitivity was abolished. These results suggest that in adult skeletal muscle fibres, the presence of a functional t-tubule network increases the sensitivity of RyR1 to agonists via a mechanism that involves binding of Ca2+ to an extracellular regulatory site.

Show MeSH
Related in: MedlinePlus