Several groups have reported alterations in ryanodine receptor (RyR) activity in response to changes in phosphorylation state. Some have shown protein kinase A (PKA)-dependent phosphorylation to increase RyR open probability (Po) in lipid bilayer experiments1,while others claim it reduces both Po and [3H]ryanodine binding to sarcoplasmic reticulum vesicles2.Similar contradictory results have been reported with Ca2+-calmodulin dependent protein kinase II3.The lack of consensus between reports has left the consequence of phosphorylation on RyR channel function poorly defined. We have therefore investigated the effects of PKA and alkaline phosphatase (AP) on the gating of sheep cardiac RYR (RyR2). We have tried to relate this to the phosphorylation state of S2809, the residue identified as the site of PKA-dependent phosphorylation on RyR21, using phosphorylation state specific antibodies4.Sarcoplasmic reticulum (SR) membrane vesicles were isolated as described previously5 from sheep hearts obtained from an abattoir and were either incorporated into artificial membranes for single channels studies or were used for [3H]ryanodine binding or Western blotting5. Western blot analysis suggests that the RyR2 channels isolated using our methods are significantly phosphorylated under basal conditions and yet we observe relatively low levels of Po in the presence of µM cytosolic Ca2+ (0.050±0.019 at 10µM free Ca2+, mean±SEM; n=7). Western blot analysis indicated a further small increase in phosphorylation at S2809 in RyR2 incubated with PKA and an increase in [3H]ryanodine binding to 294.9±68.5% (mean±SD; n=3) of control. PKA treatment of RyR2 incorporated into lipid bilayers resulted in an increase in Po from 0.114±0.045 to 0.643±0.197 (mean±SEM; n=5, T-test P<0.05). AP incubation dephosphorylated RyR2 at S2809 and caused a reduction in channel activity as demonstrated by a reduction in [3H]ryanodine binding to 20.8±7.6% (mean±SEM; n=4, T-test P<0.05) of control.Our data demonstrate that RyR2 channel activity is dependent on the phosphorylation state of S2809. However Po may remain low even in the presence of a significant level of phosphorylation at S2809. A relatively small further increment in PKA-dependent phosphorylation is required to significantly increase channel P .It appears therefore that a very high level of
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phosphorylation of RyR2 at S2809 is required to significantly increase RyR2 channel activity.