Tài liệu Báo cáo khoa học: Effect of gadolinium on the ryanodine receptor/ sarcoplasmic reticulum

Effect of gadolinium on the ryanodine receptor/

sarcoplasmic reticulum calcium release channel of skeletal

muscle

Sa´ndor Sa´rko¨ zi1

, Csaba Szegedi1

, Bala´ zs Luka´ cs1

, Michel Ronjat2 and Istva´n Jo´ na1

1 Department of Physiology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Hungary

2 Laboratoire Ioniques et Signalization, DBMS, CEA, Grenoble, France

Earlier studies revealed that lanthanide ions have sev￾eral physiological effects on different tissues and cells.

These effects seemed to be related to Ca2+-dependent

mechanisms and several studies describe direct effects

of these ions on Ca2+-binding or calcium ion conduct￾ing membrane proteins. Lanthanide ions were found to

inhibit the twitch of toad skeletal muscle with effect￾iveness depending on the ion radius. Among lanthan￾ide ions gadolinium has the strongest inhibitory effect

[1]. Gadolinum is also able to competitively displace

calcium from the sarcoplasmic reticulum (SR) [2], with

the Kd for Gd3+ in the micromolar range. On neurons,

Gd3+ blocked a part of the voltage sensitive Ca2+ cur￾rent [3]. Further studies revealed that gadolinium

blocks the voltage gated N-type [4], and T- and L-type

Ca2+ channels [5]. The inhibitory effect of Gd3+ on

SR Ca-ATPase, due to binding to the Ca-binding site,

has also been described [6]. As a result of the high

affinity of lanthanides to calcium binding sites, they

have been named as ‘supercalcium’.

Keywords

calcium channel; gadolinium; RyR1

Correspondence

I. Jo´na, 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: [email protected]

(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

[

3

H]ryanodine binding assay, 5 lm Gd3+ increased the Kd of the [3

H]ry￾anodine binding of the vesicles from 33.8 nm to 45.6 nm while Bmax, refer￾ring to the binding capacity, was not affected significantly. In the presence

of 18 nm [

3

H]ryanodine and 100 lm free Ca2+, Gd3+ inhibited the binding

of the radiolabeled ryanodine with an apparent Kd value of 14.7 lm 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 lm gadolinium. In single channel

experiments gadolinium inhibited the channel activity from both the cyto￾plasmic (cis) (IC50 ¼ 5.65 ± 0.33 lm, nHill ¼ 4.71) and the luminal (trans)

side (IC50 ¼ 5.47 ± 0.24 lm, nHill ¼ 4.31). The degree of inhibition on the

cis side didn’t show calcium dependency in the 100 lm to 1 mm Ca2+ con￾centration range which indicates no competition with calcium on its regula￾tory 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 volt￾age dependence, which would be the case if Gd3+ exerted its effect after

getting to the cis side. Our results suggest the presence of inhibitory bind￾ing sites for Gd3+ on both sides of the RyR1 with similar Hill coefficients

and IC50 values.

Abbreviations

HSR, heavy sarcoplasmic reticulum; SR, sarcoplasmic reticulum; RyR1, skeletal type ryanodine receptor.

464 FEBS Journal 272 (2005) 464–471 ª 2004 FEBS