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Effect of gadolinium on the ryanodine receptor/sarcoplasmic reticulum calcium release channel of skeletal muscle
Author(s) -
Sárközi Sándor,
Szegedi Csaba,
Lukács Balázs,
Ronjat Michel,
Jóna István
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2004.04486.x
Subject(s) - ryanodine receptor , chemistry , calcium , egta , endoplasmic reticulum , ryr1 , vesicle , biophysics , skeletal muscle , voltage dependent calcium channel , biochemistry , endocrinology , biology , membrane , organic chemistry
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, 5 µ m Gd 3+ increased the K d of the [ 3 H]ryanodine binding of the vesicles from 33.8 n m to 45.6 n m while B max , referring to the binding capacity, was not affected significantly. In the presence of 18 n m [ 3 H]ryanodine and 100 µ m free Ca 2+ , Gd 3+ inhibited the binding of the radiolabeled ryanodine with an apparent K d value of 14.7 µ m and a Hill coefficient of 3.17. In 45 Ca 2+ experiments the time constant of 45 Ca 2+ efflux from HSR vesicles increased from 90.9 (± 11.1) ms to 187.7 (± 24.9) ms in the presence of 20 µ m gadolinium. In single channel experiments gadolinium inhibited the channel activity from both the cytoplasmic ( cis ) (IC 50 = 5.65 ± 0.33 µ m , n Hill = 4.71) and the luminal ( trans ) side (IC 50 = 5.47 ± 0.24 µ m , n Hill = 4.31). The degree of inhibition on the cis side didn't show calcium dependency in the 100 µ m to 1 m m Ca 2+ concentration range which indicates no competition with calcium on its regulatory binding sites. When Gd 3+ was applied at the trans side, EGTA was present at the cis side to prevent the binding of Gd +3 to the cytoplasmic calcium binding regulatory sites of the RyR1 if Gd 3+ accidentally passed through the channel. The inhibition of the channel did not show any voltage dependence, which would be the case if Gd 3+ exerted its effect after getting to the cis side. Our results suggest the presence of inhibitory binding sites for Gd 3+ on both sides of the RyR1 with similar Hill coefficients and IC 50 values.