In vitro PKA phosphorylation of RyR2 modulates dantrolene inhibition of RyR2. (A) Representative Western blots of RyR2s isolated from sheep heart that were subjected to various incubations to manipulate RyR2 phosphorylation prior to SDS PAGE and Western blot. RyR2 were first maximally dephosphorylated by PP1 and then subjected to CaMKII phosphorylation (in the presence of PKA inhibitor H89) or PKA phosphorylation (in the presence of CaMKII inhibitor KN93). An untreated sample (neither dephosphorylated or subsequently phosphorylated, untreated) is included for comparison. Blots were probed with either antibodies for pS2808 or pS2814 and then stripped and reprobed with antibodies for RyR2. (B) Corresponding average data for RyR2 phosphorylation at S2808 and S2814. Data are normalized to the maximum phosphorylation of S2808 by PKA (left) and to S2814 by CaM (right). Bars denote mean ± SEM of seven independent experiments. Labels indicate P values for comparisons. (C) Representative single-channel recordings of RyR2 that were subjected to various incubations mentioned above. Channel recordings were taken in the presence of 100 nM cytoplasmic Ca2+ and 2 mM ATP and 100 nM CaM. Luminal [Ca2+] = 0.1 mM and membrane potential = −40 mV. Channel openings are downward current jumps from the baseline (dashed line), and arrows indicate the addition of 10 μM dantrolene. (D) RyR2 Po in the presence of 10 μM dantrolene relative to the mean of the bracketing intervals of dantrolene washout. In control experiments, RyR2 were incubated with dephosphorylation buffers lacking PP1 (PP1-ctrl) or with PP1 followed by phosphorylation buffers lacking PKA (PKA-ctrl) or lacking CaM (CaM-ctrl) but including the PKA inhibitor H89 and CaMKII inhibitor KN93. Data in bar graphs are shown as mean ± SEM. Number of samples (n) and number of experiments (N) are shown in each bar. P values denote a significant difference in relative values to 1, which are (left to right) 9 × 10−5, 2 × 10−3, 3 × 10−3, 8 × 10−9, 5 × 10−5, 0.11, and 0.03. Source data are available for this figure: SourceData F1.
Figure 1.

In vitro PKA phosphorylation of RyR2 modulates dantrolene inhibition of RyR2. (A) Representative Western blots of RyR2s isolated from sheep heart that were subjected to various incubations to manipulate RyR2 phosphorylation prior to SDS PAGE and Western blot. RyR2 were first maximally dephosphorylated by PP1 and then subjected to CaMKII phosphorylation (in the presence of PKA inhibitor H89) or PKA phosphorylation (in the presence of CaMKII inhibitor KN93). An untreated sample (neither dephosphorylated or subsequently phosphorylated, untreated) is included for comparison. Blots were probed with either antibodies for pS2808 or pS2814 and then stripped and reprobed with antibodies for RyR2. (B) Corresponding average data for RyR2 phosphorylation at S2808 and S2814. Data are normalized to the maximum phosphorylation of S2808 by PKA (left) and to S2814 by CaM (right). Bars denote mean ± SEM of seven independent experiments. Labels indicate P values for comparisons. (C) Representative single-channel recordings of RyR2 that were subjected to various incubations mentioned above. Channel recordings were taken in the presence of 100 nM cytoplasmic Ca2+ and 2 mM ATP and 100 nM CaM. Luminal [Ca2+] = 0.1 mM and membrane potential = −40 mV. Channel openings are downward current jumps from the baseline (dashed line), and arrows indicate the addition of 10 μM dantrolene. (D) RyR2 Po in the presence of 10 μM dantrolene relative to the mean of the bracketing intervals of dantrolene washout. In control experiments, RyR2 were incubated with dephosphorylation buffers lacking PP1 (PP1-ctrl) or with PP1 followed by phosphorylation buffers lacking PKA (PKA-ctrl) or lacking CaM (CaM-ctrl) but including the PKA inhibitor H89 and CaMKII inhibitor KN93. Data in bar graphs are shown as mean ± SEM. Number of samples (n) and number of experiments (N) are shown in each bar. P values denote a significant difference in relative values to 1, which are (left to right) 9 × 10−5, 2 × 10−3, 3 × 10−3, 8 × 10−9, 5 × 10−5, 0.11, and 0.03. Source data are available for this figure: SourceData F1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal