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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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Regions with re-sealed t-tubules have a higher sensitivity to RyR1 agonists. (A) A mechanically skinned fibre was field simulated in order to identify a region exhibiting Ca2+ release failure (positioned centrally in the image). Selected x–y images show the fibre under control conditions and then during the peak of responses to (i) tetanic stimulation (50 Hz for 400 ms), (ii) depolarisation of the t-tubules by substitution of K-HDTA with Na-HDTA, (iii) addition of 5 mM caffeine and (iv) 20 mM caffeine/low Mg2+. (B) Following field stimulation (50 Hz for 400 ms) to identify regions Ca2+ release failure, fibres 1–3 were exposed to a variety of stimuli, which induce a submaximal Ca2+ efflux from the SR by activating RyR1 including (1) 20 μM Mg2+, (2) 500 μM 4-CMC, or (3) 20 μM ryanodine. In each case, the submaximal stimulus was followed by a maximal response to 20 mM caffeine/low Mg2+. (C) Accumulated data illustrating the difference in caffeine sensitivity of regions responsive (filled bars) or un-responsive (open bars) to field stimulation. All responses are expressed relative to that induced by application of 20 mM caffeine/low Mg2+ in the responsive region. (*) Indicates statistically different from response in region exhibiting depolarisation-induced Ca2+ release (p < 0.05, n = 5, mean ± S.E.M.).
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fig3: Regions with re-sealed t-tubules have a higher sensitivity to RyR1 agonists. (A) A mechanically skinned fibre was field simulated in order to identify a region exhibiting Ca2+ release failure (positioned centrally in the image). Selected x–y images show the fibre under control conditions and then during the peak of responses to (i) tetanic stimulation (50 Hz for 400 ms), (ii) depolarisation of the t-tubules by substitution of K-HDTA with Na-HDTA, (iii) addition of 5 mM caffeine and (iv) 20 mM caffeine/low Mg2+. (B) Following field stimulation (50 Hz for 400 ms) to identify regions Ca2+ release failure, fibres 1–3 were exposed to a variety of stimuli, which induce a submaximal Ca2+ efflux from the SR by activating RyR1 including (1) 20 μM Mg2+, (2) 500 μM 4-CMC, or (3) 20 μM ryanodine. In each case, the submaximal stimulus was followed by a maximal response to 20 mM caffeine/low Mg2+. (C) Accumulated data illustrating the difference in caffeine sensitivity of regions responsive (filled bars) or un-responsive (open bars) to field stimulation. All responses are expressed relative to that induced by application of 20 mM caffeine/low Mg2+ in the responsive region. (*) Indicates statistically different from response in region exhibiting depolarisation-induced Ca2+ release (p < 0.05, n = 5, mean ± S.E.M.).

Mentions: The presence of readily identifiable regions of skinned fibres lacking re-sealed t-tubules was used to study the functional relationship between the DHPR/t-tubule network and associated RyRs located within the junctional SR. In Fig. 3, fibres were repeatedly field-stimulated at 50 Hz (400 ms duration) in order to identify a localised region exhibiting failure of depolarisation-induced SR Ca2+ release (not shown). In A, the Ca2+ release profile during the peak of a tetanic response to field stimulation is shown adjacent to responses induced by substitution of K-HDTA with Na-HDTA, addition of a submaximal concentration of caffeine (5 mM) or maximal activation of RyR1 induced by 20 mM caffeine/low Mg2+ (see Methods). As expected, the Ca2+-release profile in response to depolarisation of the t-tubule network following Na+ substitution was similar to that induced by field stimulation. In this cell, 2 mM caffeine failed to induce a response (not shown). A pronounced SR Ca2+ release did occur on introduction of 5 mM caffeine, but only in the regions responsive to t-tubule depolarisation. This suggests that the sensitivity of RyR1 is higher in regions of the fibre were RyRs are associated with re-sealed t-tubules. Subsequent addition of 20 mM caffeine/low Mg2+ to induce maximal activation of RyR1, resulted in a spatially homogeneous rise in [Ca2+]. Control experiments showed that the dye was not saturated during a maximal SR Ca2+ release.


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)

Regions with re-sealed t-tubules have a higher sensitivity to RyR1 agonists. (A) A mechanically skinned fibre was field simulated in order to identify a region exhibiting Ca2+ release failure (positioned centrally in the image). Selected x–y images show the fibre under control conditions and then during the peak of responses to (i) tetanic stimulation (50 Hz for 400 ms), (ii) depolarisation of the t-tubules by substitution of K-HDTA with Na-HDTA, (iii) addition of 5 mM caffeine and (iv) 20 mM caffeine/low Mg2+. (B) Following field stimulation (50 Hz for 400 ms) to identify regions Ca2+ release failure, fibres 1–3 were exposed to a variety of stimuli, which induce a submaximal Ca2+ efflux from the SR by activating RyR1 including (1) 20 μM Mg2+, (2) 500 μM 4-CMC, or (3) 20 μM ryanodine. In each case, the submaximal stimulus was followed by a maximal response to 20 mM caffeine/low Mg2+. (C) Accumulated data illustrating the difference in caffeine sensitivity of regions responsive (filled bars) or un-responsive (open bars) to field stimulation. All responses are expressed relative to that induced by application of 20 mM caffeine/low Mg2+ in the responsive region. (*) Indicates statistically different from response in region exhibiting depolarisation-induced Ca2+ release (p < 0.05, n = 5, mean ± S.E.M.).
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fig3: Regions with re-sealed t-tubules have a higher sensitivity to RyR1 agonists. (A) A mechanically skinned fibre was field simulated in order to identify a region exhibiting Ca2+ release failure (positioned centrally in the image). Selected x–y images show the fibre under control conditions and then during the peak of responses to (i) tetanic stimulation (50 Hz for 400 ms), (ii) depolarisation of the t-tubules by substitution of K-HDTA with Na-HDTA, (iii) addition of 5 mM caffeine and (iv) 20 mM caffeine/low Mg2+. (B) Following field stimulation (50 Hz for 400 ms) to identify regions Ca2+ release failure, fibres 1–3 were exposed to a variety of stimuli, which induce a submaximal Ca2+ efflux from the SR by activating RyR1 including (1) 20 μM Mg2+, (2) 500 μM 4-CMC, or (3) 20 μM ryanodine. In each case, the submaximal stimulus was followed by a maximal response to 20 mM caffeine/low Mg2+. (C) Accumulated data illustrating the difference in caffeine sensitivity of regions responsive (filled bars) or un-responsive (open bars) to field stimulation. All responses are expressed relative to that induced by application of 20 mM caffeine/low Mg2+ in the responsive region. (*) Indicates statistically different from response in region exhibiting depolarisation-induced Ca2+ release (p < 0.05, n = 5, mean ± S.E.M.).
Mentions: The presence of readily identifiable regions of skinned fibres lacking re-sealed t-tubules was used to study the functional relationship between the DHPR/t-tubule network and associated RyRs located within the junctional SR. In Fig. 3, fibres were repeatedly field-stimulated at 50 Hz (400 ms duration) in order to identify a localised region exhibiting failure of depolarisation-induced SR Ca2+ release (not shown). In A, the Ca2+ release profile during the peak of a tetanic response to field stimulation is shown adjacent to responses induced by substitution of K-HDTA with Na-HDTA, addition of a submaximal concentration of caffeine (5 mM) or maximal activation of RyR1 induced by 20 mM caffeine/low Mg2+ (see Methods). As expected, the Ca2+-release profile in response to depolarisation of the t-tubule network following Na+ substitution was similar to that induced by field stimulation. In this cell, 2 mM caffeine failed to induce a response (not shown). A pronounced SR Ca2+ release did occur on introduction of 5 mM caffeine, but only in the regions responsive to t-tubule depolarisation. This suggests that the sensitivity of RyR1 is higher in regions of the fibre were RyRs are associated with re-sealed t-tubules. Subsequent addition of 20 mM caffeine/low Mg2+ to induce maximal activation of RyR1, resulted in a spatially homogeneous rise in [Ca2+]. Control experiments showed that the dye was not saturated during a maximal SR Ca2+ release.

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