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Tài liệu Báo cáo khoa học: Effect of gadolinium on the ryanodine receptor/ sarcoplasmic reticulum calcium release channel of skeletal muscle docx

Effect of gadolinium on the ryanodine receptor/
sarcoplasmic reticulum calcium release channel of skeletal
muscle
Sa
´
ndor Sa
´
rko
¨
zi
1
, Csaba Szegedi
1
, Bala
´
zs Luka
´
cs
1
, Michel Ronjat
2

and Istva
´
nJo
´
na
1
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 Ca
2+
-dependent
mechanisms and several studies describe direct effects
of these ions on Ca
2+
-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 K
d
for Gd
3+
in the micromolar range. On neurons,
Gd
3+
blocked a part of the voltage sensitive Ca
2+
cur-
rent [3]. Further studies revealed that gadolinium
blocks the voltage gated N-type [4], and T- and L-type
Ca
2+
channels [5]. The inhibitory effect of Gd
3+
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: 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
[
3
H]ryanodine binding assay, 5 lm Gd
3+
increased the K
d
of the [
3
H]ry-
anodine binding of the vesicles from 33.8 nm to 45.6 nm while B
max
, refer-
ring to the binding capacity, was not affected significantly. In the presence
of 18 nm [
3
H]ryanodine and 100 lm free Ca
2+
,Gd
3+
inhibited the binding
of the radiolabeled ryanodine with an apparent K
d
value of 14.7 lm 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 lm gadolinium. In single channel
experiments gadolinium inhibited the channel activity from both the cyto-
plasmic (cis) (IC
50
¼ 5.65 ± 0.33 lm,n
Hill
¼ 4.71) and the luminal (trans)
side (IC
50
¼ 5.47 ± 0.24 lm,n
Hill
¼ 4.31). The degree of inhibition on the
cis side didn’t show calcium dependency in the 100 lm to 1 mm Ca
2+
con-
centration range which indicates no competition with calcium on its regula-
tory 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 volt-
age 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 bind-
ing sites for Gd
3+
on both sides of the RyR1 with similar Hill coefficients
and IC
50
values.
Abbreviations
HSR, heavy sarcoplasmic reticulum; SR, sarcoplasmic reticulum; RyR1, skeletal type ryanodine receptor.
464 FEBS Journal 272 (2005) 464–471 ª 2004 FEBS
The Ca
2+
release channel ⁄ ryanodine receptor (RyR1)
is regulated by several cytoplasmic ligands. It is activa-
ted by millimolar concentrations of ATP and micromo-
lar Ca
2+
, and inhibited by higher concentrations
(> 100 lm)ofCa
2+
and by millimolar Mg
2+
. Calmod-
ulin existing in the cytoplasm directly regulates the
channel in a Ca-dependent manner by activating the
RyR1 at [Ca
2+
]<1lm and inhibiting at higher cal-
cium concentrations [7,8]. The direct activation of the
RyR1 by cytoplasmic Ca
2+
shows a bell-shaped curve
which suggests the presence of a high-affinity activating
and a low-affinity inhibitory Ca
2+
-binding site on the
channel [8,9]. A putative high-affinity calcium-binding
site has already been found on the cytosolic site, close to
the conducting pore [10]. Results about the effect of the
luminal (trans) calcium on the activity of the RyR1 are
controversial depending on experimental conditions.
Inhibition of the channel was described at high luminal
calcium levels by Fill et al. [11] and Ma et al. [12].
Sitsapesan & Williams observed that the channel was
only regulated by the luminal calcium if it was first pre-
activated by ATP and cyclic ADP-ribose (cADPR) on
the cis side [13,14]. Tripathy et al. and Herrmann-Frank
et al. have found activation of the channel at
100–250 lm and inactivation at higher luminal calcium
concentrations [15,16]. As the effect of the luminal cal-
cium concentration was voltage-dependent and the acti-
vation depended on the cis EGTA concentration in both
cases the authors concluded that calcium ions regulate
the channel activity by binding to cytosolic Ca-binding
sites after flowing through the conducting pore. As
Gd
3+
can bind to the Ca
2+
-binding sites of different
membrane proteins we investigated the effect of this ion
on the RyR1 using different techniques [17].
Results
Single channel experiments
Figure 1 shows representative single channel records at
control state and at different concentrations of Gd
3+
applied at the cis side. During experiments gadolinium
concentration was increased successively using stock
GdCl
3
solution. Gadolinium, in the micromolar con-
centration range, decreased the probability of the
channel opening; the channel openings apparently
occured more rarely. At each concentration of gadolin-
ium the effect has developed by the end of the experi-
ment (< 30 s). At 13.3 lm Gd
3+
the RyR1 was in its
almost completely closed state (not shown). The inhibi-
tion was reversible; after adding EGTA (equimolar
with Gd
3+
) the channel almost completely regained
its original activity (not shown). As the Gd–EGTA
complex has a pK
d
of 12.6 [18], EGTA selectively
binds Gd
3+
below these circumstances. The open
probability of the channel as a function of the cis
gadolinium concentration (Fig. 2) was fitted by the
Hill equation resulting in a half-inhibitory concentra-
Fig. 1. Effect of cis gadolinium on the ryanodine receptor. Single
channel records were taken at )108 mV using symmetrical bathing
solution having 50 l
M free calcium concentration. The charge carrier
was 250 m
M KCl, and the channel openings were downward deflec-
tions. Upper panel shows the control record with a P
o
¼0.338. In
the presence of 5 l
M cis gadolinium the channel open probability
decreases to 0.148 (middle panel) while increasing the gadolinium
concentration to 8.3 l
M, the channel openings were further reduced
(lower panel) and characterized by P
o
¼ 0.019 (o represents the
open while c represents the closed state of the channel).
Fig. 2. Concentration dependence of the inhibitory effect of gado-
linium from the cis side. Single channel recordings were taken at
various cis gadolinium concentrations and open probabilities deter-
mined using 30 s record segments. Data plotted as mean ± SE.
Continuous line represents a Hill equation fitted to the data points
having the parameters of IC
50
¼ 5.65 lM and n
Hill
¼ 4.71. Number
of experiments is shown at each point.
S. Sa
´
rko
¨
zi et al. Effect of gadolinium on RyR1
FEBS Journal 272 (2005) 464–471 ª 2004 FEBS 465
tion of 5.6 ± 0.3 lm for Gd
3+
and a Hill coefficient
of 4.7 ± 0.8.
We have also tested the effect of Gd
3+
on the lumi-
nal side of the reconstituted channel. To decrease the
possibility of the applied Gd
3+
acting on the cytoplas-
mic site of the RyR1 after (or during) passing through
its conducting pore, the EGTA-Ca buffer was applied
on the cis side. The calculated free calcium concentra-
tion was equal on the cis and in the trans sides. On the
other hand, voltage step measurements were made to
see if the transmembrane potential difference had any
affect on the inhibitory action of the Gd
3+
.Gd
3+
inhibited the channel activity from the trans side in a
concentration-dependent manner. (Fig. 3A–C). Decrea-
sing the Gd
3+
concentration on the trans side of the
channel by addition of equimolar EGTA resulted in
the recovery of the channel from the inhibition
(Fig. 3D) in less than one minute. The observation
that the equimolar EGTA was able to completely
restore the channel activity from the gadolinium inhi-
bition within seconds, also strongly supports the idea
that 100 lm EGTA total in the cis side prevents the
binding of any Gd
3+
ion to the regulatory calcium-
binding sites located on the cis side of the RyR1. Fol-
lowing the recovery, the pharmacology of the RyR1
was checked by its regulators to confirm that the chan-
nel kept its integrity. Decreasing cis Ca
2+
resulted in a
decreased open probability (Fig. 3E), while successive
additions of ATP cis increased the channel activity
(Fig. 3F, first half of the record). At the end of the
experiment ryanodine was added to the cis side which
caused the channel to lock into its characteristic ‘half-
open’ state (Fig. 3F, second half of the record). The
average of the normalized open probabilities were
plotted against the trans Gd
3+
concentration (Fig. 4).
Fitting the data points with the Hill equation
gave a half-inhibitory gadolinium concentration of
5.4 ± 0.2 lm and a Hill coefficient of 4.3 ± 0.6. Open
time histograms were constructed using the same rep-
resentative current records from which the open proba-
bilities were calculated. Histograms could be fitted in a
satisfactory way by a single exponential. Representa-
tive open time constant values of the RyR1s in the
presence of Gd
3+
cis and trans are shown in Tables 1
and 2, respectively. Gadolinium decreased the open
time constant of the reconstituted ryanodine receptor
in a concentration-dependent manner acting on either
side. The decreased open time constant together with
the decreased number of the open events led to the
decrease of the open probability. The voltage-depend-
ence of the inhibitory effect of the gadolinium was also
investigated in four experiments. Gadolinium (2 lm)
was applied at the trans side of the channel and cur-
rent records were acquired at different voltage steps.
Open probability values were calculated and normal-
ized in the absence of the gadolinium. The ratios of
the normalized P
o
value to the average P
o
value during
the individual experiments were plotted against the
transmembrane potential differences (Fig. 5). Data
points could be fitted by linear regression (y ¼
Fig. 3. Effect of trans gadolinium on the ryanodine receptor. Single
channel recordings were taken as described in Materials and meth-
ods. Charge carrier was 250 mm KCl, and EGTA was applied on
the cis side in order to immediately remove gadolinium ions that
may have passed through the channel. Channel opening are down-
ward deflections. (A) shows a control record in the absence of
gadolinium characterized by P
ocontrol
¼ 0.239. Effect of gadolinium
at different concentrations are shown in (B) (4.1 l
M Gd
3+
,P
o
¼
0.147) and (C) (9 l
M Gd
3+
,Po¼ 0.032). After addition of equimolar
EGTA (9 l
M) which chelates practically the total amount of previ-
ously added gadolinium, the channel regains its original activity [(D)
calculated Gd
3+
concentration 12.3 nM,P
o
¼ 0.21]. Further addition
of EGTA to the cis side to reduce the cis Ca
2+
concentration to
0.9 l
M, results in a substantially decreased open probability (E).
The channel pharmacology is fully preserved as shown by the
effect of ATP [left side (F), 2 m
M cis ATP, 2.54 lM cis Ca
2+
] and
ryanodine [right side (F), 8 l
M ryanodine]. All records are from one
single experiment, the holding potential was )55 mV.
Effect of gadolinium on RyR1 S. Sa
´
rko
¨
zi et al.
466 FEBS Journal 272 (2005) 464–471 ª 2004 FEBS
A+BÆ X) where A and B were found to be 1.00 ±
0.07 and 0.0016 ± 0.0009, respectively, where y stands
for the corrected P
o
and X for the H
p
. As the fitting
revealed no voltage-dependence of the inhibition this
finding proves that gadolinium exerts its effect from
the luminal side of the channel and does not penetrate
to the cis side. The effect of changing cis calcium con-
centration on the inhibitory effect of cis gadolinium
was also investigated. When calcium concentration was
increased from 100 lm to 1 mm in the presence of
4.5 lm Gd
3+
cis (not shown), no significant change
was observed in the channel activity (P
o
values nor-
malized for the control were 0.011 ± 0.006 and
0.005 ± 0.002 for 100 lm and 1 mm Ca
2+
cis, respect-
ively, where the number of experiment was three)
which proves no competition beetwen Gd
3+
and Ca
2+
in this calcium contrentation range. EGTA (50 lm)
applied consecutively in the cis side resulted in a slight
but significant recovery of the channel from the inhibi-
tion to a normalized P
o
value of 0.21 ± 0.18 (n ¼ 3),
which is close to the characteristic P
o
value of the
channel at 1 mm Ca
2+
[19].
[
3
H]Ryanodine-binding and calcium efflux
The effect of gadolinium on the ADP-caffeine induced
calcium release from passively loaded HSR vesicles was
measured by the rapid filtration technique. Figure 6
shows representative data points acquired from four
representative rapid filtration experiments. The rate of
release decreased in the presence of 20 lm Gd
3+
. This
concentration of Gd
3+
increased the time taken for cal-
cium release without affecting the total amount of cal-
cium released. The effect of 5 lm gadolinium on the
[
3
H]ryanodine binding to the HSR was investigated at
different ryanodine concentrations. Gadolinium (5 lm)
Table 1. The effect of cis gadolinium on the open time constant of
RyR1.
T, Open time constant ± SE; N, number of experiments.
Gd
3+
cis
(lM) T (ms
)1
)N
0 0.99 ± 0.19 14
3.33 0.81 ± 0.13 8
5 0.57 ± 0.20 6
6.67 0.48 ± 0.05 5
Fig. 4. Concentration dependence of the inhibitory effect of gado-
linium from the trans side. Single channel recordings were taken at
various trans gadolinium concentrations and relative open probabili-
ties determined using 30 s record segments. To reduce individual
variations relative open probabilities (P
o
⁄ P
ocontrol
) at various trans
gadolinium concentrations were calculated and plotted by normal-
izing the data to the one obtained in the absence of gadolinium,
which was determined at the beginning of each experiment. Data
plotted as mean ± SE. Continuous line represents a Hill equation
fitted to the data points having the parameters of IC
50
¼ 5.47 lM
and n
Hill
¼ 4.31. Number of experiments is shown at each point.
Table 2. The effect of trans gadolinium on the open time constant
of RyR1.
T, Open time constant ± SE; N, number of experiments.
Gd
3+
trans
(lM) T (ms
)1
)N
0 1.06 ± 0.24 20
3.33 0.92 ± 0.44 6
5 0.55 ± 0.10 5
7.5 0.34 ± 0.09 3
Fig. 5. Voltage-dependence of the inhibitory effect of gadolinium
applied at the trans side. Relative (normalized for the control P
o
)
open probabilities (P
onorm
) were calculated at different transmem-
brane potential differences (± 24.8, ± 38.4, ± 56.4, ± 71.3, ± 80.2,
± 110.1 mV) in the presence of 2 l
M gadolinium trans. The ratio of
the P
onorm
values to the average of the P
onorm
values during one
experiment was plotted against the holding potential. Data points
could be satisfactory fitted by linear regression (y ¼ A+ B * x,
dashed line), where A and B were found to be 1.00 ± 0.07 and
0.0016 ± 0.0009 mV, respectively.
S. Sa
´
rko
¨
zi et al. Effect of gadolinium on RyR1
FEBS Journal 272 (2005) 464–471 ª 2004 FEBS 467
increased the dissociation constant of ryanodine-bind-
ing from 33.8 ± 3.2 to 45.6 ± 2.2 nm, while the value
of B
max
did not change significantly.
The [
3
H]ryanodine-binding assay was carried out
using HSR vesicles to study how Gd
3+
modulates the
Ca
2+
release channel ⁄ ryanodine receptor residing in
the SR membrane. Gadolinium was applied at differ-
ent concentrations in the presence of 18 nm ryanodine
and the amount of ryanodine bound was plotted
against the concentration of the gadolinium (Fig. 7).
Fitting of data points with the Hill equation revealed a
K
d
value of 14.7 ± 0.7 lm and a Hill coefficient (n
Hill
)
of 3.16 ± 0.42.
Discussion
The effect of Gd
3+
was investigated on the function of
SR Ca
2+
release channel ⁄ ryanodine receptor complex.
When the [
3
H]ryanodine-binding was examined as the
function of the gadolinium concentration we found a
K
d
value of 14.7 lm and a Hill coefficient of 3.16 for
the inhibition in the presence of 18 nm [
3
H]ryanodine.
Chevallier et al. found a K
d
value of 6.5 lm for the
La
3+
(ion which has similar properties to Gd
3+
)in
competition with calcium for calcium-binding sites of
rabbit sarcoplasmic reticulum.
RyR1s were reconstituted into an artificial lipid
bilayer and the effect of gadolinium was investigated
from the cytoplasmic and the luminal side in order to
elucidate from which side gadolinium acts. In these
single channel experiments Gd
3+
inhibited the channel
in a concentration-dependent manner on both sides.
The half-effective concentrations were about 5 lm and
the Hill coefficients were  4 for both sides. These
findings support that there are four binding sites with
similar affinity of gadolinium on the cytoplasmic and
also on the luminal side of the channel. Although in
single channel experiments the luminal Ca
2+
concen-
tration has an effect on the activity of the RyR1
[15,16], no luminal cation binding site has been des-
cribed so far. As the regulatory effect of the trans
calcium concentration was voltage-dependent and
changed as the function of the cis EGTA concentra-
tion, it was assumed that Ca
2+
goes through the chan-
nel from the luminal to the cytoplasmic side and binds
to the regulatory calcium-binding sites residing on the
cis side [16]. Several findings described in this paper
are against the possibility that the luminal Gd
3+
regu-
lates the RyR1 after getting through the channel to the
cis side. When gadolinium was applied on the trans
side (in the absence of EGTA on the trans side),
100 lm EGTA was present on the cis side which forms
a complex with the gadolinium ions if they get through
the channel. The affinity of the Gd
3+
compared to the
EGTA is a million-fold larger than that of the Ca
2+
Fig. 6. Effect of gadolinium on the calcium efflux from HSR vesi-
cles. Vesicles were loaded passively in the presence of
45
Ca. The
total calcium concentration in the loading solution was 5 m
M. Rapid
filtration experiments were carried out in quadruplets and averages
are shown. Circles represent the calcium efflux in the absence of
gadolinium while the solid line is the fitted curve assuming one
exponential component (A
max
¼ 220.0 Bq and T ¼ 73.5 ±
16.5 ms
)1
). Squares represent the calcium efflux in the presence
of 20 l
M gadolinium while the dotted line is the fitted curve assu-
ming one exponential component (A
max
¼ 215.8 Bq and T ¼
201.4 ± 25.5 ms
)1
).
Fig. 7. Gadolinium dependence of the ryanodine binding of the
channel. Ryanodine binding in the presence of 18 n
M [
3
H]ryanodine
were determined at various gadolinium concentrations. Solid line
represents the fitted curve using the Hill equation with kinetic
parameters of B
max
¼ 3.1 ± 0.1 pmolÆmg protein
)1
, K
d
¼ 14.7 ±
0.7 l
M and n
Hill
¼ 3.16 ± 0.42. Each point represents the average
of four independent measurements, while the error bars represent
SE.
Effect of gadolinium on RyR1 S. Sa
´
rko
¨
zi et al.
468 FEBS Journal 272 (2005) 464–471 ª 2004 FEBS
(K
d, Ca–EGTA
¼ 6.4, pK
d, Gd–EGTA
¼ 12.9). No voltage-
dependence of the inhibitory effect of the gadolinium
was observed in the single channel experiments (Fig. 5)
which should have happened when a cation current
flows from the trans to the cis side. The fast recovery
( 10 s) of the RyR1 from inhibition after adding
EGTA to the trans side also makes it probable that
the Gd
3+
ions were removed from luminal binding
sites of the channel. As the RyR1 displays high selec-
tivity for cations over anions [11] the relatively large
and at pH 7.2 negatively charged EGTA molecules
cannot penetrate the pore of the channel. Experiments
were carried out to investigate if there is any competi-
tion beetwen the Gd
3+
and Ca
2+
in binding to high
and low affinity calcium-binding regulatory sites of the
RyR1. In three experiments, in the presence of 4.5 lm
Gd
3+
cis, elevating free Ca
2+
concentration cis up to
1mm didn’t result in any significant change in the P
o
of the channel (0.011 ± 0.006 for 100 lm Ca
2+
and
0.005 ± 0.002 for 1 mm Ca
2+
). Consecutively applied
50 lm EGTA caused a slight recovery of the RyR1.
This concentration of EGTA decreased the [Gd
3+
]to
nominally zero whereas the calcium concentration
decreased only by some tens of lm. The calculated
open probability was characteristic for RyR1 at this
calcium concentration [19]. As the EC
50
for the cal-
cium activation of the RyR1 is 9.37 ± 1.3 [19], using
1mm calcium should have increased the activity of the
channel if competition had occured. This observation
means that cis Gd
3+
doesn’t inhibit the channel by
binding to the high affinity calcium-binding site. On
the other hand there was no further decrease in the
activity of the channel when higher cis calcium concen-
trations were applied which makes it unlikely that
gadolinium binds to the low affinity inhibitory cal-
cium-binding site. Appling calcium concentrations
higher than 1 mm would significantly worsen the signal
to noise ratio because Ca
2+
(for which the RyR1 has
much lower conductance than for K
+
) is performing a
larger role as a current carrier. Our results suggest that
there are sites on the cis and trans side of the RyR1
that are able to bind gadolinium in a reversible and
concentration-dependent manner causing the inhibition
of the channel with similar Hill coefficient and IC
50
values. The cis-binding sites seem not to be identical
with the known cis calcium-binding regulatory sites.
Materials and methods
Isolation of heavy SR vesicles
Procedure to obtain muscle was in accordance with the eth-
ical standards as formulated in the Helsinki declarations of
1975 (revised in 1983). First, HSR vesicles were isolated
from rabbit skeletal muscles (longissimus dorsii) according
to the method described by Lai & Meissner [20]. Bands cor-
responding to the HSR fractions were collected from the
36–38% region of continuous sucrose gradient, pelleted at
124 000 g for 60 min, resuspended in 0.4 m sucrose, 10 mm
K
+
⁄ PIPES, pH 7.0 and were snap frozen in liquid nitro-
gen, stored at )70 °C for ryanodine-binding and calcium-
flux measurements. Other portions of these HSR vesicles
were instantly used for ryanodine receptor purification. The
following protease inhibitors were also included throughout
the preparation procedure to prevent proteolysis during
isolation: 200 lm Pefabloc [4-(2-aminoethyl)-benzolsulfonyl-
fluoride], 100 nm aprotinin, 1 lm leupeptin, 1 lm pepstatin
A and 1 mm benzamidine. Protein concentration of HSR
vesicles was determined according to the method of Lowry
et al. [21], using bovine serum albumin as standard. Data
expressed as mean ± SE.
Purification of the ryanodine receptor complex
HSR vesicles (3 mgÆmL
)1
) were solubilized for 2 h at 4 °C
with 1% (v/v) CHAPS in a solution containing 1 m NaCl,
100 lm EGTA, 150 lm CaCl
2
,5mm AMP, 0.45% phos-
phatidylcholine, 20 mm Na ⁄ PIPES, pH 7.2, protease inhibi-
tors as in the previous step, and additionally 1 lm calpain
inhibitor I (N-acetyl-leu-leu-norleucinal) and 1 lm calpain
inhibitor II (N-acetyl-leu-leu-methionial) were also included.
The extent of ryanodine receptor solubilization and the
subsequent migration distance of the solubilized receptor in
the sucrose gradient was monitored by labeling one part of
the solubilized sample with [
3
H]ryanodine. Insoluble pro-
teins were removed by centrifugation at 59 000 g, and sub-
sequently the resulting supernatant was layered on the
top of 10–28% sucrose gradients containing 1% (v/v)
CHAPS, 0.7 m NaCl, 3.3 mm AMP, 0.5% (w/v) phosphat-
idylcholine, 70 lm EGTA, 100 lm CaCl
2
,1mm dithiothrei-
tol, 13 mm Na ⁄ PIPES, pH 7.2 and all protease inhibitors
as used before. Unlabelled and [
3
H]ryanodine-labeled solu-
bilized SR membranes were centrifuged through identical
sucrose gradients for 16 h at 90 000 g (4EC) in a swing-out
(SW-27) Beckman rotor (Beckman Inc., Fullerton, CA,
USA). Fractions of the unlabelled gradient corresponding,
by sucrose density, to the peak of the [
3
H]ryanodine-labeled
receptors were collected, rapidly frozen in liquid nitrogen
and stored at )70 °C until further use. Aliquots of the
HSR vesicles and fractions of the solubilized receptor col-
lected from the sucrose density gradient were visualized by
SDS ⁄ PAGE using Laemmli type 10% linear gel for the
visualization of the protein composition of the fractions.
Ryanodine-binding
Ryanodine-binding assays were carried out using [
3
H]ry-
anodine in a medium having the composition of 1 m NaCl,
S. Sa
´
rko
¨
zi et al. Effect of gadolinium on RyR1
FEBS Journal 272 (2005) 464–471 ª 2004 FEBS 469
25 mm Na ⁄ PIPES pH 7.1, 1 mm Pefabloc, 100 lm CaCl
2
,
resulting in about 100 lm of ionized free calcium concen-
tration. Aliquots (50 lL) containing 25 lg protein were
incubated at 37 °C for 120 min, with various concentrations
of the radioligand, as indicated in the figure legends. For
measurements of gadolinium effect, the Gd
3+
were added
30 min before the addition of tritiated ryanodine. The reac-
tion was terminated by filtering the samples on BIO-DOT
96 well filter apparatus using Millipore 50 filter paper
(Billerica, MA, USA). This was followed by washing the
dots with equal aliquots of washing medium identical to
the incubation medium except that ryanodine (hot and
cold) was omitted. Nonspecific binding was determined in
the presence of 50–100 lm ryanodine, which had been
added to the incubation mixture prior to the radioligand.
Filter papers were cut into appropriate pieces and their
radioactivity was determined using a liquid scintillation
counter (Beckman Inc.). Binding data were fitted using ori-
gin software and the Scatchard equation.
45
Ca
2+
flux measurements
HSR vesicles (1.45 mgÆmL
)1
) were passively loaded for 1 h
at room temperature in a medium containing 250 mm KCl,
20 mm K ⁄ MOPS pH 7.2, 5 mm CaCl
2
including
45
Ca
2+
.
Vesicles were spread on filter papers type: DA, pore size:
0.65 lm. Calcium efflux was initiated for time duration of
25–200 ms by the stream of measuring buffer containing
250 mm KCl, 20 mm K ⁄ MOPS, 5 mm ADP, 5 mm caffeine,
pH 7.2, with or without 20 lm GdCl
3
. The radioactivity
remaining on the filter was determined by liquid scintilla-
tion method.
Planar lipid bilayer measurements
CHAPS solubilized ryanodine receptor molecules were
incorporated into planar lipid bilayer of the Mueller–Rudin
type. The bilayer was formed across a 250 lm aperture
from a mixture of l-a-phosphatidylethanolamine, l-a-phos-
phatidylserine and l-a-phosphatidylcholine in the ratio of
5 : 4 : 1 dissolved in n-decane (20 mgÆmL
)1
). Reconstitu-
tion was initiated in symmetric solution (250 mm KCl,
50 lm free Ca
2+
,20mm PIPES, pH 7.2), when the gado-
linium was applied at the cis side. In experiments when the
effect of gadolinium from the trans side was investigated,
Ca ⁄ EGTA buffer was used at the cis side [150 lm CaCl
2
,
100 lm EGTA cis (free Ca
2+
¼ 50 lm); 50 lm Ca
2+
with-
out EGTA trans]. Small aliquots of the solubilized receptor
were added to one side of the bilayer chamber defined as
the cytoplasmic (cis) side, meanwhile the other chamber
was regarded as luminal (trans) side. Measurements were
carried out under voltage clamp conditions, the holding
potential (H
p
) was referred to the trans chamber as a
ground. Successful incorporation was detected as stepwise
increase in current. Electrical signals were filtered at 1 kHz
through an eight pole low-pass Bessel filter and digitized at
3 kHz, using Axon Instruments (Union City, CA, USA)
hardware and pclamp 6Æ3 software. Channels with conduct-
ance higher than 460 pS were accepted as RyR1s. Open
probability values (P
o
) were calculated from representative
data segments of 10–90 s duration. Total recording time in
each experiment was more than 5 min for the experimental
condition tested. This time duration appeared to be enough
to reach the equilibrium of the channel parameters. Tem-
perature of the bilayer chamber was 22–25 °C during meas-
urements and concentration values of ionized (‘free’) Ca
2+
were calculated using the computer program and binding
constants published by Fabiato [22].
Materials
Protease inhibitors were purchased from Boehringer
(Mannheim, Germany), from Merck (Darmstadt, Germany)
and from Sigma (St. Louis, MO, USA), lipids were
obtained from Avanti Polar Lipids (Alabaster, AL, USA),
[
3
H]ryanodine was from DUPONT (Boston, MA, USA),
SDS, polyacrylamide gel components, and molecular mass
standards were from Bio-Rad Laboratories (Hercules, CA,
USA). All other chemicals were from Sigma.
Acknowledgements
This work was supported by a grant form the Hungar-
ian Research Found (OTKA T 037727 and TS 040773),
by the Hungarian Ministry of Health (015 ⁄ 2001) and by
the European Union (HPRN-CT-2002-00331). We also
thank Prof. Varda Shoshan-Barmatz of Ben Gurion
University for her valuable help and suggestions in the
experimental design and evaluation. Cs. Szegedi is a
Boyai Fellow.
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