REGULATION OF RYANODINE RECEPTOR CALCIUM-RELEASE CHANNELS BY DIADENOSINE POLYPHOSPHATES

[H-3]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca2+ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects o f endogenous compounds termed diadenosine polyphosphates (Ap(n)As; n = 2-6 phosphate groups) on [H-3]ryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic stren gth buffer conditions, [H-3] ryanodine binding to brain membranes was significantly increased by 171% with 333 mu M P-1,P-5-di (adenosine-5' ) pentaphosphate (Ap(5)A) and by 209% with the same concentration of t he metabolism-resistant ATP analogue beta gamma-methyleneadenosine 5'- triphosphate (AMP-PCP) compared with control values for [H-3] ryanodin e binding of 9.6 +/- 1.8 fmol/mg of protein. Dose-related increases in [H-3]ryanodine binding were observed for all five Ap(n)As tested [P-1 ,P-2-di(adenosine-5') pyrophosphate (Ap(2)A), P-1,P-3-di(adenosine-5') triphosphate (Ap(3)A), P-1,P-4-di (adenosine-5') tetraphosphate (Ap(4 )A), Ap(5)A, and P-1,P-6-di (adenosine-5') hexaphosphate (Ap(6)A)] as well as AMP-PCP; oxidized salts of Ap(n)As stimulated [H-3]ryanodine b inding to a greater degree than did nonoxidized Ap(n)As. The apparent rank order for the capacity of these agents to increase [H-3]-ryanodin e binding was oxidized Ap(4)A = oxidized Ap(5)A > oxidized Ap(3)A > Ap (6)A > AMP-PCP > Ap(5)A > Ap(2)A. Addition of the approximate EC(50) d ose of oxidized Ap(4)A (37 mu M) increased the affinity (K-D) of ryano dine receptors from 34 +/- 7 to 12 +/- 2 nM; the apparent binding site density (B-max) was not significantly different from control values o f 107 +/- 33 fmol/mg of protein. Increases in [H-3]-ryanodine binding by either oxidized Ap(4)A or nonoxidized Ap(5)A were not further enhan ced by coincubation with AMP-PCP, which suggests a similar site of act ion for the Ap,As and AMP-PCP. [H-3] Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized Ap(4)A, Ap(5)A, and AMP-PCP. Oxidized Ap(4)A increased the specific binding b y ninefold in skeletal muscle and by threefold in cardiac muscle. Thes e results suggest that Ap(n)As, at physiologically relevant concentrat ions, may serve as endogenous modulators of ryanodine receptor-gated C a2+ release channels.