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1.
Fig. 8.

Fig. 8. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Ryr1D/D muscle fibers exhibit pronounced summation of the Ca2+ transient during repetitive stimulation. A: Fluo-4 transient responses to single AP stimuli followed by a 100-Hz train of APs show reduced amplitude of Ca2+ twitch and tetanic transients in mutant fibers. B: traces from A normalized to initial transient amplitude to demonstrate greater summation of the Ca2+ transient during repetitive stimulation of mutant fibers when compared with controls. C: quantification of Ca2+ transient summation from B, calculated as the ratio of the amplitude of the last Ca2+ transient in the train compared with the first. Summation was increased approximately twofold in the mutant fibers. *P < 0.01.

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
2.
Fig. 6.

Fig. 6. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Biochemical properties of membrane fractions isolated from Ryr1+/+ and Ryr1D/D mice. A: maximal specific binding capacity (Bmax) values of [3H]ryanodine binding to crude skeletal muscle membrane fractions of 1.5- to 5.5-mo-old Ryr1+/+ and Ryr1D/D mice were determined as described in experimental procedures. Normalized data are means ± SE of 4–6 experiments. *P < 0.05 compared with Ryr1+/+ mice. B: 45Ca2+ uptake rates by crude membrane fractions of 1.5- to 5.5-mo-old Ryr1+/+ and Ryr1D/D mice were determined as described in experimental procedures. Normalized data are means ± SE of 4–5 experiments.

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
3.
Fig. 11.

Fig. 11. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Ryr1D/D mice exhibit decreased twitch force generation and increased force summation in vivo. A: representative twitch (single AP) and tetanic (100 Hz, 200 ms) force responses from the tibialis anterior muscles of anesthetized Ryr1+/+ and Ryr1D/D mice. B: force vs. frequency of stimulation relationships for Ryr1+/+ (n = 5) and Ryr1D/D (n = 6) mice. C: specific twitch force of Ryr1+/+ and Ryr1D/D mice. D: specific tetanic force of Ryr1+/+ and Ryr1D/D mice. E: tetanus-to-twitch ratio, an indicator of summation, of Ryr1+/+ and Ryr1D/D mice. *P < 0.05

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
4.
Fig. 3.

Fig. 3. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

CaM regulation of RyR1 from skeletal muscle of Ryr1+/+ and Ryr1D/D mice. Specific [3H]ryanodine binding to crude membrane fractions from skeletal muscle of Ryr1+/+ (•) and Ryr1D/D (○) mice was determined at the indicated CaM concentrations in the presence of 0.15 μM Ca2+ and 1 mM AMPPCP (a nonhydrolyzable ATP analog) (A) and 25 μM (B) free Ca2+ as described in experimental procedures. Data are means ± SE of 4 experiments. *P < 0.05 compared with control (−CaM).

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
5.
Fig. 5.

Fig. 5. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Effects of S100A1 on single Ryr1+/+ and Ryr1D/D channel activities at 0.2 μM Ca2+. Membranes isolated from skeletal muscle of Ryr1+/+ (left) and Ryr1D/D (right) mice were fused with lipid bilayer. Representative single-channel currents (downward deflections from closed levels, c–) were recorded at −35 mV at 0.2 μM cis, cytosolic free Ca2+ in the presence of 1 mM ATP as described in experimental procedures in the absence of S100A1 (top traces) and following the addition of 0.3 μM (middle traces) and 1 μM (bottom traces) S100A1 to the cis chamber. Normalized Po (bottom) was obtained from 2-min recordings by setting the threshold level at 25% (open bars) and 50% (gray bars) of the current amplitude between the closed (c) and open (o) channel states. Data are means ± SE of 3–14 channel recordings. *P < 0.05 compared with control (−S100A1).

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
6.
Fig. 10.

Fig. 10. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Ryr1D/D muscle fibers exhibit reduced Ca2+ release flux with slowed inactivation of Ca2+ release in response to voltage-clamp depolarizations. A: average Ca2+ release flux from control (left; n = 6) and Ryr1D/D (middle; n = 8) fibers calculated from Fluo-4 fluorescent transients elicited by steplike 80-ms depolarizations from a holding potential of −80 mV. At right is the release vs. voltage (R-V) relationships of control and Ryr1D/D fibers. SR Ca2+ release flux was significantly suppressed in mutant muscle fibers. *P < 0.05. B: Ca2+ release flux from A elicited by depolarizing pulses to −20, 0, and +20 mV from a holding potential of −80 mV, normalized to peak flux to investigate the relative time course of Ca2+ release. While the time course of release was similar between mutant and control fibers at lesser depolarizations (−20 mV, left), upon larger depolarizations (0 mV, +20 mV), mutant fibers demonstrate less rapid inactivation of release compared with controls, as seen by the pronounced shoulder during the fast inactivation of release flux in Ryr1D/D fibers.

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
7.
Fig. 7.

Fig. 7. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Ryr1D/D muscle fibers exhibit normal Indo-1 resting ratio but decreased peak amplitude of the Indo-1 Ca2+ transient following a single action potential (AP). A: average Indo-1 ratio Ca2+ transients from Ryr1+/+ (black trace; n = 20) and Ryr1D/D (gray trace; n = 22) fibers. Isolated fibers were stimulated with field electrodes at 400 ms, and emission ratio was examined. B: bar plot summarizing resting Indo-1 ratio averages (left) and peak ΔIndo-1 ratio (right) of control and mutant fibers. No significant (NS) differences in resting Ca2+ concentration were detected, but a significant difference in peak ΔIndo-1 ratio was found (*P = 0.017). C: averaged Indo-1 ratio responses from control (black traces; n = 12) and mutant (gray traces; n = 12) fibers elicited with a two-AP protocol with recovery periods of 40, 80, and 160 ms between stimuli. Traces were normalized to amplitude of the first stimulus to appreciate relative summation in mutant fibers.

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
8.
Fig. 1.

Fig. 1. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Generation of mice with an L3625D mutation in the calmodulin (CaM)-binding site of ryanodine receptor 1 (RyR1). A: schematic representation of the mouse Ryr1 gene and targeting construct. S and N represent SpeI and NdeI restriction enzyme sites, respectively. neo, tACE-cre, and TK denote neomycin-resistant gene, Cre recombinase gene driven by testis-specific angiotensin-converting enzyme promoter, and thymidine kinase gene, respectively. Arrows and “x” indicate the position of primers for screening and a mutation site, respectively. B: southern blot analysis of genomic DNA. After restriction enzyme digestion of genomic DNA with SpeI and NdeI, 5′- and 3′-probes identify 12.2-kb and 5.1-kb fragment in targeted allele, respectively. Both probes hybridize with 16.7-kb fragments in wild-type (WT) allele. C: sequence analysis of RT-PCR. cDNA encoding CaM-binding site of RyR1 was amplified from total RNA from homozygous (hybrid genetic background) mouse skeletal muscle and sequenced. The L3625D mutation was confirmed. A HinfI site created by the L3625D mutation (GATTC) was used for screening the mutant allele.

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
9.
Fig. 9.

Fig. 9. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Ryr1D/D muscle fibers demonstrate summation of AP-evoked Ca2+ transients and slower inactivation of sarcoplasmic reticulum (SR) Ca2+ release flux during tetanic stimulation. A: average Fluo-4 transient responses of 7 control and 10 mutant fibers to a single AP stimuli followed by 5 repeats of 200 ms trains of 100 Hz stimuli, with 200 ms recovery between trains. Traces are normalized to the amplitude of the first transient to demonstrate differences in transient summation during repetitive stimulation. B, left: boxed traces from A, time expanded and normalized to amplitude of the initial peak to examine transient summation of last train. Right: quantification of transient facilitation between Ryr1+/+ and Ryr1D/D fibers (*P < 0.01, Ryr1+/+ n = 7, Ryr1D/D n = 10). C: average SR Ca2+ release flux of control and mutant fibers, quantified using a Ca2+ removal model to calculate release from fluorescence responses of fibers subjected to stimulation paradigm seen in A. Release flux is suppressed in mutant fibers compared with controls. D: representative SR Ca2+ release flux time course of first tetanic train in C from a control and mutant fiber, normalized to amplitude of initial flux. Peaks of release flux are fit to a single exponential function to quantify inactivation of release during the train. Tau of inactivation is significantly increased in Ryr1D/D fibers, suggesting a slower rate of inactivation. Fractional inactivation was not significantly different between the groups.

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
10.
Fig. 2.

Fig. 2. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

Effects of CaM on single Ryr1+/+ and Ryr1D/D channel activities. Membranes isolated from skeletal muscle of Ryr1+/+ and Ryr1D/D mice were fused with lipid bilayer as described in experimental procedures. Representative single-channel currents (downward deflections from closed levels, c–) were recorded at −35 mV at 0.2 μM (A) and 7 μM (B) cis, cytosolic free Ca2+ in the presence of 1 mM ATP as described in experimental procedures in the absence of CaM (top traces) and following the addition of 20 nM (middle traces) and 200 nM (bottom traces) cis CaM. Channel open probabilities (Po) obtained from 2-min recordings in the absence of CaM were 0.15 ± 0.02 (n = 21) and 0.14 ± 0.02 (n = 29) at 50% threshold setting, 0.30 ± 0.04 and 0.23 ± 0.02 at 25% threshold setting at 0.2 μM Ca2+ and 1 mM ATP, and 0.27 ± 0.05 (n = 14) and 0.26 ± 0.05 (n = 11) at 50% threshold setting and 0.38 ± 0.05 and 0.48 ± 0.05 at 25% threshold setting at 7 μM Ca2+ and 1 mM ATP for Ryr1+/+ and Ryr1D/D, respectively. Normalized Po values (bottom) were obtained from 2-min recordings by setting the threshold level at 25% (open bars) and 50% (gray bars) of the current amplitude between the closed (c) and open (o) channel states. Data are means ± SE of 9 and 15 (A) and 14 and 11 (B) channel recordings for Ryr1+/+ and Ryr1D/D, respectively. *P < 0.05 compared with respective control (−CaM).

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.
11.
Fig. 4.

Fig. 4. From: Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

CaCaM and CaS100A1 bound to the CaM/S100A1-binding domain of RyR1. A: space-filling model of dimeric CaS100A1 with each subunit bound to a peptide (RyRP12; shown in red) from the CaM/S100A1-binding domain of RyR1. In the foreground, one subunit is shown illustrating the location of RyR1 residues W3621 and L3625 on the hydrophobic side of an amphipathic helix, which in turn interact with hydrophobic residues on CaS100A1 (shown in blue). B: space-filling diagram of CaCaM bound to a peptide (red) from the CaM/S100A1-binding domain of RyR1 illustrating that these two residues (W3621, L3625) also interact directly with hydrophobic residues on CaCaM (shown in blue). C: ribbon diagram illustrating a close-up view of the CaS100A1-RyRP12 interaction. D: ribbon diagram illustrating a close-up view of the CaCaM-RyR1 peptide interaction. In AD, calcium ions are colored orange. E: binding of CaS100A1 to WT and mutant RyRP12 peptides derived from RyR1 as determined using isothermal titration calorimetry (ITC). Shown is a representative trace together with a theoretical fitted curve for the binding of the WT-RyRP12 peptide to CaS100A1 (▴). These data were duplicated to provide the thermodynamic binding parameters (n = 0.75 ± 0.12; KD = 75 ± 12 μM; ΔH = −5.8 ± 0.6 kcal/mol). Also shown are representative data for the binding of the RyR1-L3625D mutant peptide together with a theoretical curve for binding. In this case, the binding stoichiometry could not be quantitatively determined since the binding was weak and was assumed to be one (n = 1) in all the titrations measured; therefore, these data provided only a limiting value for binding (□ KD > 0.9 ± 0.1 mM). No detectable binding was observed for the RyR1-W3621D (•) or the RyR1-W3621D + L3625D [double mutant (DM); ◊] mutant peptides.

Naohiro Yamaguchi, et al. Am J Physiol Cell Physiol. 2011 May;300(5):C998-C1012.

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