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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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Localised Ca2+ release failure occurs where t-tubules have not re-sealed. A skinned EDL fibre was exposed to a solution containing 20 μM fluo-3 acid for 20 min. This resulted in a progressive accumulation of dye within re-sealed t-tubules and vesicular elements on the left of the x–y image, but not on the right (a). In the continued presence of fluo-3, the preparation was field stimulated (50 Hz for 400 ms) and the resulting rise in cytosolic [Ca2+] occurred in a region of the cell exhibiting dye accumulation within the re-sealed t-tubules (b). Application of saponin caused a transient rise in [Ca2+] in the same region of the cell (c) and more prolonged exposure (2 min) resulted in loss of dye trapped within the re-sealed t-tubules (d). After saponin treatment, field stimulation failed to elicit a response (e). Subtraction of the fluo-3 fluorescence remaining after saponin treatment (i.e. cytocolic + bound) from the fluorescence in the control image (i.e. cytosolic + bound + t-tubule) provides a corrected image showing the dye localised within the saponin-sensitive compartments (f). All responses were obtained in the same fibre. Corrected image (d) re-scaled to enhance contrast and clearly identify the region with dye trapped in the t-tubule.
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fig2: Localised Ca2+ release failure occurs where t-tubules have not re-sealed. A skinned EDL fibre was exposed to a solution containing 20 μM fluo-3 acid for 20 min. This resulted in a progressive accumulation of dye within re-sealed t-tubules and vesicular elements on the left of the x–y image, but not on the right (a). In the continued presence of fluo-3, the preparation was field stimulated (50 Hz for 400 ms) and the resulting rise in cytosolic [Ca2+] occurred in a region of the cell exhibiting dye accumulation within the re-sealed t-tubules (b). Application of saponin caused a transient rise in [Ca2+] in the same region of the cell (c) and more prolonged exposure (2 min) resulted in loss of dye trapped within the re-sealed t-tubules (d). After saponin treatment, field stimulation failed to elicit a response (e). Subtraction of the fluo-3 fluorescence remaining after saponin treatment (i.e. cytocolic + bound) from the fluorescence in the control image (i.e. cytosolic + bound + t-tubule) provides a corrected image showing the dye localised within the saponin-sensitive compartments (f). All responses were obtained in the same fibre. Corrected image (d) re-scaled to enhance contrast and clearly identify the region with dye trapped in the t-tubule.

Mentions: The absence of depolarisation-induced SR Ca2+ release in localised regions of mechanically skinned fibres (Fig. 1C) might be explained by failure of the t-tubules to re-seal. This possibility was investigated using the protocol shown in Fig. 2, where a skinned fibre was exposed to 20 μM fluo-3 for 20 min, resulting in accumulation of the dye in the t-tubules (see Methods). A region exhibiting a characteristic striated pattern due to t-tubule dye localization [15,19] was positioned on the left of the x–y frame, while a darker region, apparently lacking trapped dye, occupied the remainder of the field (Fig. 2a). As described in previous studies, the re-sealed region also contained dye trapped within sarcolemmal vesicles, which appear as bright spherical or longitudinal elements [19].


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)

Localised Ca2+ release failure occurs where t-tubules have not re-sealed. A skinned EDL fibre was exposed to a solution containing 20 μM fluo-3 acid for 20 min. This resulted in a progressive accumulation of dye within re-sealed t-tubules and vesicular elements on the left of the x–y image, but not on the right (a). In the continued presence of fluo-3, the preparation was field stimulated (50 Hz for 400 ms) and the resulting rise in cytosolic [Ca2+] occurred in a region of the cell exhibiting dye accumulation within the re-sealed t-tubules (b). Application of saponin caused a transient rise in [Ca2+] in the same region of the cell (c) and more prolonged exposure (2 min) resulted in loss of dye trapped within the re-sealed t-tubules (d). After saponin treatment, field stimulation failed to elicit a response (e). Subtraction of the fluo-3 fluorescence remaining after saponin treatment (i.e. cytocolic + bound) from the fluorescence in the control image (i.e. cytosolic + bound + t-tubule) provides a corrected image showing the dye localised within the saponin-sensitive compartments (f). All responses were obtained in the same fibre. Corrected image (d) re-scaled to enhance contrast and clearly identify the region with dye trapped in the t-tubule.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Localised Ca2+ release failure occurs where t-tubules have not re-sealed. A skinned EDL fibre was exposed to a solution containing 20 μM fluo-3 acid for 20 min. This resulted in a progressive accumulation of dye within re-sealed t-tubules and vesicular elements on the left of the x–y image, but not on the right (a). In the continued presence of fluo-3, the preparation was field stimulated (50 Hz for 400 ms) and the resulting rise in cytosolic [Ca2+] occurred in a region of the cell exhibiting dye accumulation within the re-sealed t-tubules (b). Application of saponin caused a transient rise in [Ca2+] in the same region of the cell (c) and more prolonged exposure (2 min) resulted in loss of dye trapped within the re-sealed t-tubules (d). After saponin treatment, field stimulation failed to elicit a response (e). Subtraction of the fluo-3 fluorescence remaining after saponin treatment (i.e. cytocolic + bound) from the fluorescence in the control image (i.e. cytosolic + bound + t-tubule) provides a corrected image showing the dye localised within the saponin-sensitive compartments (f). All responses were obtained in the same fibre. Corrected image (d) re-scaled to enhance contrast and clearly identify the region with dye trapped in the t-tubule.
Mentions: The absence of depolarisation-induced SR Ca2+ release in localised regions of mechanically skinned fibres (Fig. 1C) might be explained by failure of the t-tubules to re-seal. This possibility was investigated using the protocol shown in Fig. 2, where a skinned fibre was exposed to 20 μM fluo-3 for 20 min, resulting in accumulation of the dye in the t-tubules (see Methods). A region exhibiting a characteristic striated pattern due to t-tubule dye localization [15,19] was positioned on the left of the x–y frame, while a darker region, apparently lacking trapped dye, occupied the remainder of the field (Fig. 2a). As described in previous studies, the re-sealed region also contained dye trapped within sarcolemmal vesicles, which appear as bright spherical or longitudinal elements [19].

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