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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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Spontaneous localised Ca2+ release events in sealed and unsealed regions. (A) Longitudinal line scan images from two mechanically skinned fibres. In the first cell, approximately half of the scan line was positioned in a region responsive to field stimulation, while the other half was positioned in an adjacent unresponsive region (upper panel). Ca2+ sparks and more prolonged “embers” are apparent, but most are restricted to the responsive region. A fibre exhibiting spontaneous SR Ca2+ release events is also shown (lower panel). Again, spontaneous Ca2+ release events are restricted to the region exhibiting spontaneous release events. (B) Longitudinal line scan images from a mechanically skinned fibre in the presence of either K-HDTA (upper panel) or Na-HDTA (lower panel). In the presence of K-HDTA spontaneous Ca2+ release events are apparent and the Ca2+ transient in response to field stimulation confirms that the t-tubules are polarised and functional throughout the image. On substitution of Na-HDTA with K-HDTA, both the spontaneous Ca2+ release events and the response to field stimulation were abolished. (C) Cumulative data obtained from four fibres exhibiting localised Ca2+ release events. In each cell, half of the longitudinal scan line was positioned in a region responsive to field stimulation and the other half in a region unresponsive to field stimulation. The ordinate shows the total number of spontaneous Ca2+ release events in four fibres at the position indicated either before (black bars) or after depolarisation of the t-tubule network with Na-HDTA (open bars). All distances are relative to the boundary between adjacent responsive and unresponsive regions of the cell. (*) Indicates significantly greater number of events than in adjacent unresponsive region (p < 0.05, n = 4, mean ± S.E.M.). ns indicates not significantly different from adjacent unresponsive region.
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fig6: Spontaneous localised Ca2+ release events in sealed and unsealed regions. (A) Longitudinal line scan images from two mechanically skinned fibres. In the first cell, approximately half of the scan line was positioned in a region responsive to field stimulation, while the other half was positioned in an adjacent unresponsive region (upper panel). Ca2+ sparks and more prolonged “embers” are apparent, but most are restricted to the responsive region. A fibre exhibiting spontaneous SR Ca2+ release events is also shown (lower panel). Again, spontaneous Ca2+ release events are restricted to the region exhibiting spontaneous release events. (B) Longitudinal line scan images from a mechanically skinned fibre in the presence of either K-HDTA (upper panel) or Na-HDTA (lower panel). In the presence of K-HDTA spontaneous Ca2+ release events are apparent and the Ca2+ transient in response to field stimulation confirms that the t-tubules are polarised and functional throughout the image. On substitution of Na-HDTA with K-HDTA, both the spontaneous Ca2+ release events and the response to field stimulation were abolished. (C) Cumulative data obtained from four fibres exhibiting localised Ca2+ release events. In each cell, half of the longitudinal scan line was positioned in a region responsive to field stimulation and the other half in a region unresponsive to field stimulation. The ordinate shows the total number of spontaneous Ca2+ release events in four fibres at the position indicated either before (black bars) or after depolarisation of the t-tubule network with Na-HDTA (open bars). All distances are relative to the boundary between adjacent responsive and unresponsive regions of the cell. (*) Indicates significantly greater number of events than in adjacent unresponsive region (p < 0.05, n = 4, mean ± S.E.M.). ns indicates not significantly different from adjacent unresponsive region.

Mentions: Fig. 6A, shows a recording in line-scan mode from a mechanically skinned fibre, with the scan line positioned longitudinally through adjacent regions, which were either responsive, or non-responsive to field stimulation. A single 2 ms stimulus resulted in a brief twitch response and a corresponding rise in cytosolic Ca2+, which was limited to the area of the cell in the lower half of the image (left). While quiescent, the cell also exhibited Ca2+ sparks and longer embers, similar to those reported in intact and skinned skeletal cells. However, these events were predominantly restricted to the region of the cell responsive to field stimulation. As described previously [11], in the absence of cytocolic Cl−, which normally serves to stabilize the membrane potential, some skinned cells exhibit spontaneous depolarisations, resulting in repeated twitch-like responses. An example of this is shown in Fig. 6B, where large Ca2+ transients arose spontaneously at irregular intervals. In this case the upper half of the scan line was positioned through a region producing spontaneous Ca2+ transients, while the lower half was quiescent and insensitive to field stimulation (not shown). Again, spontaneous Ca2+ sparks were apparent, but only at high frequency in the region of the fibre exhibiting spontaneous Ca2+ transients.


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)

Spontaneous localised Ca2+ release events in sealed and unsealed regions. (A) Longitudinal line scan images from two mechanically skinned fibres. In the first cell, approximately half of the scan line was positioned in a region responsive to field stimulation, while the other half was positioned in an adjacent unresponsive region (upper panel). Ca2+ sparks and more prolonged “embers” are apparent, but most are restricted to the responsive region. A fibre exhibiting spontaneous SR Ca2+ release events is also shown (lower panel). Again, spontaneous Ca2+ release events are restricted to the region exhibiting spontaneous release events. (B) Longitudinal line scan images from a mechanically skinned fibre in the presence of either K-HDTA (upper panel) or Na-HDTA (lower panel). In the presence of K-HDTA spontaneous Ca2+ release events are apparent and the Ca2+ transient in response to field stimulation confirms that the t-tubules are polarised and functional throughout the image. On substitution of Na-HDTA with K-HDTA, both the spontaneous Ca2+ release events and the response to field stimulation were abolished. (C) Cumulative data obtained from four fibres exhibiting localised Ca2+ release events. In each cell, half of the longitudinal scan line was positioned in a region responsive to field stimulation and the other half in a region unresponsive to field stimulation. The ordinate shows the total number of spontaneous Ca2+ release events in four fibres at the position indicated either before (black bars) or after depolarisation of the t-tubule network with Na-HDTA (open bars). All distances are relative to the boundary between adjacent responsive and unresponsive regions of the cell. (*) Indicates significantly greater number of events than in adjacent unresponsive region (p < 0.05, n = 4, mean ± S.E.M.). ns indicates not significantly different from adjacent unresponsive region.
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fig6: Spontaneous localised Ca2+ release events in sealed and unsealed regions. (A) Longitudinal line scan images from two mechanically skinned fibres. In the first cell, approximately half of the scan line was positioned in a region responsive to field stimulation, while the other half was positioned in an adjacent unresponsive region (upper panel). Ca2+ sparks and more prolonged “embers” are apparent, but most are restricted to the responsive region. A fibre exhibiting spontaneous SR Ca2+ release events is also shown (lower panel). Again, spontaneous Ca2+ release events are restricted to the region exhibiting spontaneous release events. (B) Longitudinal line scan images from a mechanically skinned fibre in the presence of either K-HDTA (upper panel) or Na-HDTA (lower panel). In the presence of K-HDTA spontaneous Ca2+ release events are apparent and the Ca2+ transient in response to field stimulation confirms that the t-tubules are polarised and functional throughout the image. On substitution of Na-HDTA with K-HDTA, both the spontaneous Ca2+ release events and the response to field stimulation were abolished. (C) Cumulative data obtained from four fibres exhibiting localised Ca2+ release events. In each cell, half of the longitudinal scan line was positioned in a region responsive to field stimulation and the other half in a region unresponsive to field stimulation. The ordinate shows the total number of spontaneous Ca2+ release events in four fibres at the position indicated either before (black bars) or after depolarisation of the t-tubule network with Na-HDTA (open bars). All distances are relative to the boundary between adjacent responsive and unresponsive regions of the cell. (*) Indicates significantly greater number of events than in adjacent unresponsive region (p < 0.05, n = 4, mean ± S.E.M.). ns indicates not significantly different from adjacent unresponsive region.
Mentions: Fig. 6A, shows a recording in line-scan mode from a mechanically skinned fibre, with the scan line positioned longitudinally through adjacent regions, which were either responsive, or non-responsive to field stimulation. A single 2 ms stimulus resulted in a brief twitch response and a corresponding rise in cytosolic Ca2+, which was limited to the area of the cell in the lower half of the image (left). While quiescent, the cell also exhibited Ca2+ sparks and longer embers, similar to those reported in intact and skinned skeletal cells. However, these events were predominantly restricted to the region of the cell responsive to field stimulation. As described previously [11], in the absence of cytocolic Cl−, which normally serves to stabilize the membrane potential, some skinned cells exhibit spontaneous depolarisations, resulting in repeated twitch-like responses. An example of this is shown in Fig. 6B, where large Ca2+ transients arose spontaneously at irregular intervals. In this case the upper half of the scan line was positioned through a region producing spontaneous Ca2+ transients, while the lower half was quiescent and insensitive to field stimulation (not shown). Again, spontaneous Ca2+ sparks were apparent, but only at high frequency in the region of the fibre exhibiting spontaneous Ca2+ transients.

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