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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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Patchy Ca2+ release persists after depolarisation of the t-tubules. A mechanically skinned fibre was initially perfused with a solution containing K-HDTA and field-stimulated in order to identify a region exhibiting SR Ca2+ release failure (upper panel). Selected x–y images are shown under control conditions (left) and during the peaks of responses to tetanic field stimulation (middle) or 5 mM caffeine (right). The same fibre was then exposed to a solution in which K-HDTA was substituted with Na-HDTA to induce a sustained depolarisation of the t-tubules (lower panel). The initial exposure to Na-HDTA induced a localised rise in Ca2+ due to depolarisation of the t-tubule network (not shown). In the continued presence of Na-HDTA, field stimulation failed to elicit a response (left). On addition of 5 mM caffeine, the rise in [Ca2+] occurred in the region of the cell responsive to field stimulation (middle). Subsequent addition of 20 mM caffeine/low Mg2+ induced a uniform Ca2+ release.
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fig4: Patchy Ca2+ release persists after depolarisation of the t-tubules. A mechanically skinned fibre was initially perfused with a solution containing K-HDTA and field-stimulated in order to identify a region exhibiting SR Ca2+ release failure (upper panel). Selected x–y images are shown under control conditions (left) and during the peaks of responses to tetanic field stimulation (middle) or 5 mM caffeine (right). The same fibre was then exposed to a solution in which K-HDTA was substituted with Na-HDTA to induce a sustained depolarisation of the t-tubules (lower panel). The initial exposure to Na-HDTA induced a localised rise in Ca2+ due to depolarisation of the t-tubule network (not shown). In the continued presence of Na-HDTA, field stimulation failed to elicit a response (left). On addition of 5 mM caffeine, the rise in [Ca2+] occurred in the region of the cell responsive to field stimulation (middle). Subsequent addition of 20 mM caffeine/low Mg2+ induced a uniform Ca2+ release.

Mentions: The role of t-tubule polarisation was investigated using the protocol shown in Fig. 4. A fibre was initially field-stimulated and regions lacking SR Ca2+ release identified (upper panel). As in previous examples, only the region exhibiting depolarisation-induced Ca2+ release (middle) responded to 5 mM caffeine (right). The same fibre was then perfused with a solution in which K-HDTA was substituted with Na-HDTA in order to induce sustained depolarisation of the t-tubules (lower panel). Following Na-substitution, the fibre was unresponsive to field stimulation, consistent with sustained depolarisation of the t-tubule network (lower, left). However, on subsequent addition of 5 mM caffeine, the pattern of Ca2+ release remained similar to that observed under control conditions (lower, middle). This suggests that the higher sensitivity to caffeine in regions responsive to field stimulation is unrelated to the polarisation status of the re-sealed t-tubules. Again, addition of 20 mM caffeine/low Mg2+ induced a uniform release throughout the fibre (lower, right), demonstrating that RyRs remain functional throughout the preparation and that the SR [Ca2+] content is relatively uniform. Similar results were obtained in five 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)

Patchy Ca2+ release persists after depolarisation of the t-tubules. A mechanically skinned fibre was initially perfused with a solution containing K-HDTA and field-stimulated in order to identify a region exhibiting SR Ca2+ release failure (upper panel). Selected x–y images are shown under control conditions (left) and during the peaks of responses to tetanic field stimulation (middle) or 5 mM caffeine (right). The same fibre was then exposed to a solution in which K-HDTA was substituted with Na-HDTA to induce a sustained depolarisation of the t-tubules (lower panel). The initial exposure to Na-HDTA induced a localised rise in Ca2+ due to depolarisation of the t-tubule network (not shown). In the continued presence of Na-HDTA, field stimulation failed to elicit a response (left). On addition of 5 mM caffeine, the rise in [Ca2+] occurred in the region of the cell responsive to field stimulation (middle). Subsequent addition of 20 mM caffeine/low Mg2+ induced a uniform Ca2+ release.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571965&req=5

fig4: Patchy Ca2+ release persists after depolarisation of the t-tubules. A mechanically skinned fibre was initially perfused with a solution containing K-HDTA and field-stimulated in order to identify a region exhibiting SR Ca2+ release failure (upper panel). Selected x–y images are shown under control conditions (left) and during the peaks of responses to tetanic field stimulation (middle) or 5 mM caffeine (right). The same fibre was then exposed to a solution in which K-HDTA was substituted with Na-HDTA to induce a sustained depolarisation of the t-tubules (lower panel). The initial exposure to Na-HDTA induced a localised rise in Ca2+ due to depolarisation of the t-tubule network (not shown). In the continued presence of Na-HDTA, field stimulation failed to elicit a response (left). On addition of 5 mM caffeine, the rise in [Ca2+] occurred in the region of the cell responsive to field stimulation (middle). Subsequent addition of 20 mM caffeine/low Mg2+ induced a uniform Ca2+ release.
Mentions: The role of t-tubule polarisation was investigated using the protocol shown in Fig. 4. A fibre was initially field-stimulated and regions lacking SR Ca2+ release identified (upper panel). As in previous examples, only the region exhibiting depolarisation-induced Ca2+ release (middle) responded to 5 mM caffeine (right). The same fibre was then perfused with a solution in which K-HDTA was substituted with Na-HDTA in order to induce sustained depolarisation of the t-tubules (lower panel). Following Na-substitution, the fibre was unresponsive to field stimulation, consistent with sustained depolarisation of the t-tubule network (lower, left). However, on subsequent addition of 5 mM caffeine, the pattern of Ca2+ release remained similar to that observed under control conditions (lower, middle). This suggests that the higher sensitivity to caffeine in regions responsive to field stimulation is unrelated to the polarisation status of the re-sealed t-tubules. Again, addition of 20 mM caffeine/low Mg2+ induced a uniform release throughout the fibre (lower, right), demonstrating that RyRs remain functional throughout the preparation and that the SR [Ca2+] content is relatively uniform. Similar results were obtained in five 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