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The effect of gadolinium ions on the sarcoplasmic reticulum (SR) calcium release channel⁄ryanodine receptor (RyR1) was studied using heavy SR (HSR) vesicles and RyR1 isolated from rabbit fast twitch muscle. In the [ 3 H]ryanodine binding assay, 5lmGd 3+ increased the Kd of the [ 3 H]ry-anodine binding of the vesicles from 33.8 nmto 45.6 nmwhileBmax, refer-ring to the binding capacity, was not affected significantly. In the presence of 18 nm[ 3 H]ryanodine and 100lmfree Ca 2+ ,Gd 3+ inhibited the binding of the radiolabeled ryanodine with an apparentKd value of 14.7lmand a Hill coefficient of 3.17. . | iFEBS Journal Effect of gadolinium on the ryanodine receptor sarcoplasmic reticulum calcium release channel of skeletal muscle Sandor Sarkozi1 Csaba Szegedi1 Balazs Lukács1 Michel Ronjat2 and Istvan Jona1 1 Department of Physiology Research Center for Molecular Medicine Medicaland Health Science Center University of Debrecen Hungary 2 Laboratoire Ioniques et Signalization DBMS CEA Grenoble France Keywords calcium channel gadolinium RyR1 Correspondence I. Jona Department of Physiology Research Center for Molecular Medicine Medical and Health Science Center University of Debrecen Debrecen H-4012 Hungary Fax 36 52 432289 Tel 36 52 416634 E-mail jonai@phys.dote.hu Received 10 September 2004 revised 10 November 2004 accepted 16 November 2004 doi 10.1111 j.1742-4658.2004.04486.x The effect of gadolinium ions on the sarcoplasmic reticulum SR calcium release channel ryanodine receptor RyR1 was studied using heavy SR HSR vesicles and RyR1 isolated from rabbit fast twitch muscle. In the 3H ryanodine binding assay 5 pM Gd3 increased the Kd of the 3H ry-anodine binding of the vesicles from 33.8 nM to 45.6 nM while Bmax referring to the binding capacity was not affected significantly. In the presence of 18 nM 3H ryanodine and 100 IM free Ca2 Gd3 inhibited the binding of the radiolabeled ryanodine with an apparent Kd value of 14.7 IM and a Hill coefficient of 3.17. In 45Ca2 experiments the time constant of 45Ca2 efflux from HSR vesicles increased from 90.9 11.1 ms to 187.7 24.9 ms in the presence of 20 IM gadolinium. In single channel experiments gadolinium inhibited the channel activity from both the cytoplasmic cis IC50 5.65 0.33 pM nHill 4.71 and the luminal trans side IC50 5.47 0.24 pM nHill 4.31 . The degree of inhibition on the cis side didn t show calcium dependency in the 100 IM to 1 mM Ca2 concentration range which indicates no competition with calcium on its regulatory binding sites. When Gd3 was applied at the trans side EGTA was present at the cis side to prevent the
