MODULATION OF CARDIAC RYANODINE RECEPTORS OF SWINE AND RABBIT BY A PHOSPHORYLATION-DEPHOSPHORYLATION MECHANISM

1. The regulation of the cardiac Ca2+ release channel-ryanodine recept or (RYR) by exogenous acid phosphatase (AcPh) and purified Ca2+-calmod ulin-dependent protein kinase II (CaMKII) was studied in swine and rab bit sarcoplasmic reticulum (SR) vesicles using [H-3]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1- 20 U ml(-1)) to a standard incubation medium increased [H-3]ryanodine binding in a Ca2+-dependent manner. Stimulation was only readily appar ent in media containing micromolar Ca2+ concentrations. 3. Scatchard a nalysis of [H-3]ryanodine binding curves revealed that AcPh enhanced b inding by increasing the affinity of the receptor for [H-3]ryanodine w ithout recruiting additional receptor sites (K-d, 9.8 +/- 0.85 and 3.9 +/- 0.65 nM; B-max (the maximal receptor density), 1.45 +/- 0.14 and 1.47 +/- 0.12 pmol mg(-1) for control and AcPh, respectively). The fai lure of AcPh to increase B-max suggested that the number of receptors that were 'dormant' due to phosphorylation in the SR preparation was v ery small. 4. At the single channel level, AcPh increased the open pro bability (P-o) of RyR channels by increasing the opening rate and indu cing the appearance of a longer open state while having no effect on s ingle channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [H- 3]ryanodine binding and P-o of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a syn thetic peptide inhibitor of CaMIKII, or replacement of ATP with the no n-hydrolysable ATP analogue adenyly[beta gamma-methylene]-diphosphate (AMP-PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibi tion of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation-dephosphorylati on mechanism effectively regulates RyRs. 7. The results suggest that i ntracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activi ty. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2.