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+ accidenta
Trang 1sarcoplasmic reticulum calcium release channel of skeletal muscle
Sa´ndor Sa´rko¨zi1, Csaba Szegedi1, Bala´zs Luka´cs1, Michel Ronjat2and 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 Kdfor 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: 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 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 [3H]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) (IC50¼ 5.65 ± 0.33 lm, nHill¼ 4.71) and the luminal (trans) side (IC50¼ 5.47 ± 0.24 lm, nHill¼ 4.31) The degree of inhibition on the cisside 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 IC50values
Abbreviations
HSR, heavy sarcoplasmic reticulum; SR, sarcoplasmic reticulum; RyR1, skeletal type ryanodine receptor.
Trang 2The Ca2+release channel⁄ ryanodine receptor (RyR1)
is regulated by several cytoplasmic ligands It is
activa-ted by millimolar concentrations of ATP and
micromo-lar Ca2+, and inhibited by higher concentrations
(> 100 lm) of Ca2+and by millimolar Mg2+
Calmod-ulin existing in the cytoplasm directly regulates the
channel in a Ca-dependent manner by activating the
RyR1 at [Ca2+] < 1 lm and inhibiting at higher
cal-cium concentrations [7,8] The direct activation of the
RyR1 by cytoplasmic Ca2+shows a bell-shaped curve
which suggests the presence of a high-affinity activating
and a low-affinity inhibitory Ca2+-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
Gd3+ can bind to the Ca2+-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 Gd3+
applied at the cis side During experiments gadolinium
concentration was increased successively using stock
GdCl3 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 Gd3+the RyR1 was in its
almost completely closed state (not shown) The
inhibi-tion was reversible; after adding EGTA (equimolar
with Gd3+) the channel almost completely regained
its original activity (not shown) As the Gd–EGTA
complex has a pKd of 12.6 [18], EGTA selectively binds Gd3+ 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 Po¼0.338 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 l M and n Hill ¼ 4.71 Number
of experiments is shown at each point.
Trang 3tion of 5.6 ± 0.3 lm for Gd3+ and a Hill coefficient
of 4.7 ± 0.8
We have also tested the effect of Gd3+ on the
lumi-nal side of the reconstituted channel To decrease the
possibility of the applied Gd3+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 Gd3+ Gd3+
inhibited the channel activity from the trans side in a
concentration-dependent manner (Fig 3A–C)
Decrea-sing the Gd3+ 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 Gd3+ 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 Ca2+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 Gd3+ 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 Gd3+ cisand 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 diffecur-rent voltage steps Open probability values were calculated and normal-ized in the absence of the gadolinium The ratios of the normalized Povalue to the average Povalue during the individual experiments were plotted against the transmembrane potential differences (Fig 5) Data points could be fitted by linear regression (y¼
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 Pocontrol¼ 0.239 Effect of gadolinium
at different concentrations are shown in (B) (4.1 l M Gd 3+ , Po¼ 0.147) and (C) (9 l M Gd 3+ , Po ¼ 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 n M , Po¼ 0.21] Further addition
of EGTA to the cis side to reduce the cis Ca2+ 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 l M cis Ca2+] and ryanodine [right side (F), 8 l M ryanodine] All records are from one single experiment, the holding potential was )55 mV.
Trang 4A + B Æ 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 Po and X for the Hp 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 Gd3+ cis (not shown), no significant change was observed in the channel activity (Po values nor-malized for the control were 0.011 ± 0.006 and 0.005 ± 0.002 for 100 lm and 1 mm Ca2+cis, respect-ively, where the number of experiment was three) which proves no competition beetwen Gd3+and Ca2+
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 Povalue of 0.21 ± 0.18 (n ¼ 3), which is close to the characteristic Po value of the channel at 1 mm Ca2+[19]
[3H]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 Gd3+ This concentration of Gd3+increased the time taken for cium release without affecting the total amount of cal-cium released The effect of 5 lm gadolinium on the [3H]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+
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 (Po⁄ 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 l M
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.
Fig 5 Voltage-dependence of the inhibitory effect of gadolinium applied at the trans side Relative (normalized for the control Po) open probabilities (Ponorm) 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 Ponormvalues to the average of the Ponormvalues 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.
Trang 5increased the dissociation constant of
ryanodine-bind-ing from 33.8 ± 3.2 to 45.6 ± 2.2 nm, while the value
of Bmaxdid not change significantly
The [3H]ryanodine-binding assay was carried out
using HSR vesicles to study how Gd3+ modulates the
Ca2+ release channel⁄ ryanodine receptor residing in
the SR membrane Gadolinium was applied at
differ-ent concdiffer-entrations 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
Kdvalue of 14.7 ± 0.7 lm and a Hill coefficient (nHill)
of 3.16 ± 0.42
Discussion
The effect of Gd3+was investigated on the function of
SR Ca2+ release channel⁄ ryanodine receptor complex
When the [3H]ryanodine-binding was examined as the
function of the gadolinium concentration we found a
Kd value of 14.7 lm and a Hill coefficient of 3.16 for
the inhibition in the presence of 18 nm [3H]ryanodine
Chevallier et al found a Kd value of 6.5 lm for the
La3+ (ion which has similar properties to Gd3+) 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 Gd3+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 Ca2+ 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 Ca2+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 Gd3+ 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 Gd3+compared to the EGTA is a million-fold larger than that of the Ca2+
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 Bmax¼ 3.1 ± 0.1 pmolÆmg protein)1, Kd¼ 14.7 ± 0.7 l M and nHill¼ 3.16 ± 0.42 Each point represents the average
of four independent measurements, while the error bars represent SE.
Trang 6(Kd, Ca–EGTA¼ 6.4, pKd, Gd–EGTA¼ 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 Gd3+ 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 Gd3+ and Ca2+ in binding to high
and low affinity calcium-binding regulatory sites of the
RyR1 In three experiments, in the presence of 4.5 lm
Gd3+ cis, elevating free Ca2+ concentration cis up to
1 mm didn’t result in any significant change in the Po
of the channel (0.011 ± 0.006 for 100 lm Ca2+ and
0.005 ± 0.002 for 1 mm Ca2+) Consecutively applied
50 lm EGTA caused a slight recovery of the RyR1
This concentration of EGTA decreased the [Gd3+] 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 EC50 for the
cal-cium activation of the RyR1 is 9.37 ± 1.3 [19], using
1 mm calcium should have increased the activity of the
channel if competition had occured This observation
means that cis Gd3+ 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 Ca2+ (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 IC50
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 4C with 1% (v/v) CHAPS in a solution containing 1 m NaCl,
100 lm EGTA, 150 lm CaCl2, 5 mm 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 [3H]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 CaCl2, 1 mm dithiothrei-tol, 13 mm Na⁄ PIPES, pH 7.2 and all protease inhibitors
as used before Unlabelled and [3H]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 [3H]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,
Trang 725 mm Na⁄ PIPES pH 7.1, 1 mm Pefabloc, 100 lm CaCl2,
resulting in about 100 lm of ionized free calcium
concen-tration Aliquots (50 lL) containing 25 lg protein were
incubated at 37C for 120 min, with various concentrations
of the radioligand, as indicated in the figure legends For
measurements of gadolinium effect, the Gd3+ 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-ginsoftware and the Scatchard equation
45Ca2+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 CaCl2 including 45Ca2+
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 GdCl3 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 soluReconstitu-tion (250 mm KCl,
50 lm free Ca2+, 20 mm 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 CaCl2,
100 lm EGTA cis (free Ca2+¼ 50 lm); 50 lm Ca2+
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 (Hp) 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 (Po) 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 22–25C during meas-urements and concentration values of ionized (‘free’) Ca2+
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), [3H]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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