Effect of gadolinium on the ryanodine receptor/sarcoplasmic reticulum calcium release channel of skeletal muscle
2005
Sárközi, Sándor | Szegedi, Csaba | Lukács, Balázs | Ronjat, Michel | Jóna, István
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 µm Gd³⁺ increased the Kd of the [³H]ryanodine 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 µm free Ca²⁺, Gd³⁺ inhibited the binding of the radiolabeled ryanodine with an apparent Kd value of 14.7 µm 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 µm gadolinium. In single channel experiments gadolinium inhibited the channel activity from both the cytoplasmic (cis) (IC₅₀ = 5.65 ± 0.33 µm, nHill = 4.71) and the luminal (trans) side (IC₅₀ = 5.47 ± 0.24 µm, nHill = 4.31). The degree of inhibition on the cis side didn't show calcium dependency in the 100 µm 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 binding of Gd⁺³ to the cytoplasmic calcium binding regulatory sites of the RyR1 if Gd³⁺ accidentally passed through the channel. The inhibition of the channel did not show any voltage dependence, which would be the case if Gd³⁺ exerted its effect after getting to the cis side. Our results suggest the presence of inhibitory binding sites for Gd³⁺ on both sides of the RyR1 with similar Hill coefficients and IC₅₀ values.
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