(Received for publication, August 26, 1994; and in revised form, November 3, 1994)
From the
FKBP-12 (FKBP), the soluble receptor for the immunosuppresant
drug FK-506, is tightly bound to the calcium release channel
(CRC)/ryanodine receptor (RyR) of skeletal muscle terminal cisternae
(TC) of sarcoplasmic reticulum with a stoichiometry of 4 mol of FKBP
per tetrameric RyR complex. FKBP displays cis/trans-peptidyl-prolyl isomerase (PPIase) activity
which is inhibited by FK-590 or rapamycin. In skeletal muscle TC,
FK-590 or rapamycin binds to and dissociates FKBP from the RyR in a
time- and temperature-dependent manner which increases the open
probability of the channel. Therefore, the net energized Ca uptake rate of TC vesicles devoid of FKBP is reduced due to the
increased leak of Ca
from the TC specifically via the
RyR, which is reversed upon rebinding of FKBP. Thus, the RyR is
modulated by FKBP (Timerman, A. P., Ogunbumni, E., Freund, E. A.,
Wiederrecht, G., Marks, A. R., and Fleischer, S.(1993) J. Biol.
Chem. 268, 22922-22999; Mayrleitner, M., Timerman, A. P.,
Wiederrecht, G., and Fleischer S.(1994) Cell Calcium 15,
99-108). We now find that FKBP can be displaced from the
FKBP
RyR complex by exchange with FKBP in solution. The EC
for exchange is 0.30 µM for wild type FKBP versus 0.6 to 2.4 µM for three different
site-directed mutants that are practically devoid of any measurable
PPIase activity. Substitution of wild-type FKBP on the RyR complex with
these PPIase-deficient mutants did not alter the Ca
flux of TC vesicles, whereas dissociation of FKBP from TC with
FK-590 increased the Ca
leak rate. Our studies show
that, in vivo, the FKBP
RyR complex is in equilibrium
with the cytosolic pool of FKBP (
3 µM) and suggest
that modulation of the CRC by FKBP is independent of PPIase activity.
The intracellular calcium release channels are a new class of
channels characterized by their large size and 4-fold symmetry. Their
importance is in intracellular signaling via Ca.
There are two types, the ryanodine receptor (RyR) (
)and
inositol 1,4,5-trisphosphate receptor. The ryanodine receptors from
skeletal muscle (isoform 1, RyR-1) and heart (isoform 2, RyR-2) are
involved in Ca
release in excitation-contraction
coupling (Fleischer and Inui, 1989).
Most of the ryanodine receptor
extends out from the junctional face membrane of the terminal cisternae
of SR into the myoplasm. In skeletal muscle, the ryanodine receptor is
in junctional contact with the transverse tubule to form the triad
junction. The receptor is activated to release Ca from the SR by sensing the depolarization of the transverse
tubule. The ryanodine receptor or calcium release channel (CRC) has now
been extensively characterized and has been cloned and expressed. (For
reviews, see Coronado et al. (1994), Fleischer and Inui(1989),
and McPherson and Campbell(1993).) The mass of the native CRC is 2.3
million daltons (Saito et al., 1989) which is, by far, the
largest ion channel complex known. Image enhancement analysis of
electron micrographs of the purified CRC reveals a striking 4-fold
symmetry (Wagenknecht et al., 1989). Until recently, the
native CRC was considered to be a homotetramer, consisting of four
565,000-dalton protomers (Takeshima et al., 1989; Zorzatto et al., 1990).
Recently, FKBP has been found to be tightly
associated with the ryanodine receptor (Jayaraman et al.,
1992). FKBP, the soluble receptor for the immunosuppressive drug
FK-506, is a polypeptide of 12 kDa found in the cytosol of most
eukaryotic cells. The discovery of the association of FKBP with the RyR
derives from our cloning and sequencing studies (Marks et al.,
1989, 1990). Peptide sensitivity mapping was carried out on the
purified skeletal muscle CRC. Twenty-four peptides were generated and
located on the surface of the CRC (Marks et al., 1990). One
additional peptide was isolated (Marks et al., 1989) which was
not part of the primary sequence of the ryanodine receptor protomer
(Takeshima et al., 1989) and was not in Genbank at that time.
A later search identified this peptide as the N-terminal sequence of
FKBP-12 (Collins, 1991). The CRC from skeletal muscle TC was then shown
to contain tightly bound FKBP (Jayaraman et al., 1992). The
stoichiometry of FKBPRyR receptor was found to be 4. Thus, the
native CRC of skeletal muscle SR is a heterooligomer consisting of four
ryanodine receptor protomers and 4 molecules of FKBP. Its chemical
formula can therefore be represented as (FKBP)
/(RyR
protomer)
(Timerman et al., 1993).
FK-506 and
cyclosporin A are potent immunosuppressant drugs used to prevent graft
rejection following organ transplantation. Although structurally
unrelated, these drugs block the same calcium-dependent mechanism
necessary for transcription of the early lymphokine genes required for
T-lymphocyte activation (Sigal and Dumont, 1992). FK-506 and
cyclosporin A are specific ligands for two distinct families of
soluble, intracellular receptors (immunophilins) termed FK-506 binding
proteins (FKBP) (Siekierka et al., 1989) and cyclophilins
(CyP) (Handschumaker et al., 1984), respectively. The
immunophilins possess cis/trans-peptidyl-prolyl
isomerase activity (PPIase or rotamase); that is, they catalyze the cis/trans isomerization about peptidyl-proline bonds (Harding et al., 1989; Siekierka et al., 1989). Although the
PPIase activity of each immunophilin is inhibited by its specific drug,
inhibition of rotamase activity is unrelated to the immunosuppressive
action of the drugs. Instead, the action of these drugs results from
the inhibition of calcineurin, a calmodulin-dependent/calcium-activated
protein phosphatase, by the FKBPFK-506 or cyclophilin-cyclosporin
A complexes (Liu et al., 1991). The inhibition of calcineurin
blocks translocation into the nucleus of the cytosolic component of
NF-AT, a key transcription factor required for activation of
T-lymphocytes (Schreiber and Crabtree, 1992).
The finding that the
CRC is tightly associated with FKBP provides a system to study the role
of this immunophilin in skeletal muscle E-C coupling. We found that the
characteristics of the CRC were profoundly altered by removal of FKBP
and restored to normal by its rebinding. These studies provided the
first evidence of a physiological role for FKBP (Mayrleitner et
al., 1994; Timerman et al., 1993). FKBP regulates the
CRC, by altering the sensitivity of the channel to Ca (Mayrleitner et al., 1994) and/or Mg
(this study) and stabilizes the closed state. Our conclusion is
supported by a recent study on the single channel characteristics of
the cloned skeletal muscle RyR expressed in Sf9 cells in the presence
and absence of FKBP (Brillantes et al., 1994). In this study,
we examine the possible role of PPIase activity by FKBP on the function
of the CRC by replacing bound FKBP with site-directed mutants of FKBP
which are practically devoid of PPIase activity.
With regard to the net
calcium loading rate assay (see later), preincubation of TC with TSK
column buffer up to 10% (v/v) enhances the net calcium loading rate of
TC in the cuvette assay, while incubation with 20% (v/v) or more TSK
buffer reduced the net loading rate. In order to achieve final FKBP
concentrations of up to 30 µM FKBP without exceeding an
acceptable level of TSK column buffer, several of the recombinant FKBP
preparations were further concentrated on a Centricon 3 ultrafiltration
device (Amicon) and diluted with PS buffer (20 mM NaPO, pH 6.8, containing 100 mM NaCl) to
final protein concentrations ranging from 3.0 to 6.5 mg/ml. Recombinant
FKBP preparations isolated at higher concentrations were diluted 5- to
10-fold into PS buffer to final concentrations of 3 to 5 mg/ml. For all
experiments in this study, TC vesicles were diluted from stocks of 7.5
to 15 mg of protein/ml in imidazole homogenization medium (IHM; 5
mM imidazole chloride, pH 7.4, 0.3 M sucrose, and 1
µg/ml leupeptin) to 2.5 mg of protein/ml in IHM containing 5% (v/v)
TSK column buffer.
Figure 2:
Concentration dependence for exchange of
soluble [S]FKBP-12 with bound FKBP-12. A, exchange (binding) was performed by incubation of TC
(
,
) or LT (
,
) at 2.5 mg/ml in IHM buffer
containing 0.1 to 3.0 µM [
S]FKBP-12, as indicated, for 20 min at 37
°C. Exchange isotherms are plotted for both total (
,
)
and nonspecific binding (
,
), as indicated. Nonspecific
binding was estimated by adding 10-fold excess unlabeled FKBP-12 at
each concentration of [
S]FKBP-12. B,
Scatchard analysis of the specific (i.e. total minus
nonspecific) binding to TC derived from the experiment shown. The mean
``binding parameters'' obtained from four other TC
preparations by Scatchard analysis of exchange isotherms from 0.1 to
1.5 µM [
S]FKBP-12 were: App K
(``apparent'' dissociation
constant) = 0.33 ± 0.16 µM; App B
(``apparent'' B
)
= 95.6 ± 28 pmol/mg of TC; and r (correlation
coefficient) = 0.905 ± 0.007 (n =
4).
Displacement of bound
[S]FKBP from prelabeled TC by dissociation with
FKBP ligands (such as FK-590) or by ``re-exchange'' with
unlabeled wild type or mutant FKBP preparations was performed by
incubation of prelabeled vesicles (2.5 mg/ml) at 37 °C for 20 min.
Following incubation, the amount of [
S]FKBP
bound to the SR, in this and all other experiments, was determined as
follows: 1) a 20- or 40-µl sample of SR (50 or 100 µg of
protein) was diluted into 200 µl of ice cold IHM buffer and
immediately sedimented in a Beckman TL100.1 rotor at 95,000 rpm for 15
min at 4 °C to separate bound from free
[
S]FKBP; 2) the pellets were rinsed with 200
µl of deionized water (room temperature) before resuspension of the
pellet with a second addition of 200 µl of deionized water; 3)
S bound to the pellet was determined by liquid
scintillation counting in 4.5 ml of Cytoscint (ICN).
We examined the effect of PPIase-deficient FKBP mutants to restore the net calcium loading rate of both drug-treated and FKBP-stripped TC. While assessment of rebinding (i.e. reconstitution) by each recombinant protein is more straightforward for FKBP-stripped TC (see below), the assessment of function (loading) is more complicated. For this reason, both untreated and drug-treated samples were sedimented in the same rotor to minimize handling error. Despite this precaution, small variations due to sample handling were still detectable. Therefore, the experiments shown in the results are only those performed with drug-treated TC, which eliminates one step of sample handling.
For
FKBP-stripped TC, the interpretation of this post-labeling protocol is
straightforward. For drug-treated TC, the probability that a
significant percentage of endogenous FKBP that was dissociated by
binding FK-590 rebinds to TC under these conditions is unlikely since
1) in the cold, added [S]FKBP (or unlabeled
FKBP) binds rapidly to drug-treated TC (less than 3 min), yet the
exchange reaction is negligible; 2) recombinant FKBPs are added in 10-
to 20-fold excess over endogenous FKBP. Therefore, the concentration of
endogenous FKBP is no more than 5-10% of the total FKBP; and 3)
the FKBP
drug complex does not bind to the RyR, and the endogenous
wild-type FKBP has a higher affinity for FK-590 than the
PPIase-deficient mutants used in these experiments (Table 1).
Thus, the wild-type FKBP in the main remains in the supernatant
complexed with FK-590. Western blot analysis of drug-treated TC,
reconstituted with recombinant fusion proteins (for example GST/FKBP),
which can be distinguished from wild-type FKBP by SDS-polyacrylamide
gel electrophoresis confirms that little (if any) endogenous FKBP
rebinds to TC under these conditions (not shown). Therefore, this
post-labeling protocol provides a rapid assay to assess reconstitution
of drug-treated TC with mutant FKBPs. The more complete the
reconstitution with unlabeled FKBP, the less
[
S]FKBP is bound in the postlabeling assay (Table 4).
Calcium
loading rates were obtained in a medium containing either low (3 to 4
mM) or high (10 mM) Mg. In low
Mg
buffer, the rate was calculated for several pulses
of 12.5 µM calcium. Since the loading rate is sometimes
reduced with each additional pulse (as shown in Fig. 7), the uptake
rates were calculated by averaging the rates from the first three
pulses of calcium. In high Mg
loading medium, the
rate was determined in response to a single pulse of 50 µM calcium. With regard to the effect of FK-590 on the calcium
loading rate of TC, similar results were obtained by the two different
loading rate protocols, i.e. drug-treated TC consistently have
about 60% of the net calcium loading rate of either untreated TC or
drug-control TC.
The 50% effective concentration (EC)
values obtained for either the calcium loading rates and/or
[
S]FKBP displacement from prelabeled TC in Fig. 3, 5A, and 5B were generated by fitting
the data to the generalized ligand binding equation: y = (a)/[1 + (x/b)
] by nonlinear regression using
SigmaPlot (Marquart-Levenberg algorithm), where a represents
the 100% (or full range of) activity or binding values, b is
the EC
value, and c represents the slope response
where a value of 1 indicates a normal, hyperbolic response (Limbird,
1986).
Figure 3:
Exchange
of FKBP mutants with [S]FKBP on prelabeled TC.
Prelabeled TC (88 pmol of [
S]FKBP per mg of
protein) were incubated with graded concentrations of unlabeled
wild-type or mutant FKBP for 20 min at 37 °C; the amount of
[
S]FKBP remaining in the pellet was then
determined following sedimentation of the TC in a Beckman TL100.1 rotor
as described under ``Experimental Procedures.'' The unlabeled
recombinant proteins used in this experiment include (from left to right): Y82F (
), wild-type FKBP (
), F36Y
(
), L30A (
), W59H (
), F99Y (
), and Y26F
(
). The EC
values obtained from two experiments
for each FKBP preparation is summarized in Table 1.
The time and temperature dependence for exchange of bound
FKBP is presented in Fig. 1. The exchange rate is greatly
accelerated by increasing the temperature from 0 °C to 37 °C.
At 0-4 °C, 15-20% of the total
[S]FKBP-12 binding sites are filled instantly
with little additional binding or exchange thereafter for up to 2 h.
Thus, the rate of exchange for soluble [
S]FKBP
is very slow at 0-4 °C, perhaps a few percent in 2 h. At 37
°C, the half-time (t
) to saturate the
remaining binding sites (i.e. those that are not rapidly
filled at 0-4 °C) is less than 5 min, while at 22 °C the t
is greater than 1 h. The temperature effect on
the kinetics of the exchange reaction resembles that observed for
[
H]FK-816 binding/dissociation of bound FKBP-12
(Timerman et al., 1993).
Figure 1:
Time and temperature
dependence for exchange of bound FKBP-12 by
[S]FKBP-12. The time course for total binding (i.e. exchange) of [
S]FKBP-12 to TC
vesicles (2.5 mg/ml) was measured in IHM buffer containing 2 µM [
S]FKBP-12 at either 37 °C (
),
ambient temperature (22 °C,
), or on ice (0 to 4 °C,
). The reaction was quenched at the indicated time point by
diluting an aliquot of 20 µl (50 µg of TC) into 200 µl of
ice-cold IHM buffer. The amount of bound
[
S]FKBP-12 was determined by liquid
scintillation counting of the TC following the separation of free from
bound [
S]FKBP-12 by sedimentation in a Beckman
TL 100.1 rotor as described under ``Experimental
Procedures.'' Each data point represents the average ± S.D.
obtained from four different TC
preparations.
The concentration of
[S]FKBP binding sites in TC vesicles was
determined from [
S]FKBP concentration (exchange)
isotherms. The concentration of RyR (i.e. the specific FKBP
binding site) in LT fractions is only 10% of that in TC preparations
and is due to contamination of the LT fraction with TC (Saito et
al., 1984). Thus, the LT serve as negative control (see Fig. 2A). Total [
S]FKBP binding
(exchange) to TC is saturated with increasing concentrations of
[
S]FKBP in a hyperbolic fashion (Fig. 2A). Treatment of these data by classical
equilibrium binding (Scatchard) analysis of a ligand (i.e. [
S]FKBP) to its receptor (i.e. the
RyR) yields a straight line (Fig. 2B) consistent with a
single class of [
S]FKBP binding sites in TC with
an apparent dissociation constant (K
) of 0.33
± 0.16 µM [
S]FKBP (see Fig. 2legend). The apparent B
value of
95.6 ± 28 pmol of [
S]FKBP binding sites
per mg of TC derived from Scatchard analysis ( Fig. 2legend) can
be adjusted with the following correction factors: 1) 1.25, which
corrects for only 80% saturation of the binding sites during the 20-min
incubation at 37 °C (see Fig. 1), and 2) 1.1, which corrects
for the overestimation of nonspecific binding in the presence of only
10-fold excess unlabeled FKBP (see ``Experimental
Procedures''); therefore, the value for specific binding is low by
about 10% or 10 pmol of FKBP/mg of TC. The combined correction factor
of 1.35 yields a calculated B
of 129 pmol of
[
S]FKBP binding sites per mg of TC (95.6 pmol/mg
1.35). The B
values for
[
H]FK-816 and [
H]ryanodine
binding to these same TC preparations is 124 and 28 pmol per mg of TC,
respectively (see Table 1in Timerman et al.(1993)). In
summary, the B
value for
[
S]FKBP binding (exchange) is essentially the
same as the B
value for drug
([
H]FK-816) binding in TC. Since there is only
one high affinity [
H]ryanodine binding site per
tetrameric RyR, the stoichiometry is about 4 mol of FKBP per CRC, by
either method.
The labeling of terminal cisternae by
[S]FKBP ( Fig. 1and Fig. 2) was
suggestive of an exchange process since labeling was saturable with
concentration, yet the B
value (Fig. 2B) was essentially the same as that obtained by
ligand binding ([
H]FK-816) isotherms. The
experiments in Fig. 1and Fig. 2were repeated, in the
presence of 0.25 µM soluble
[
S]FKBP, i.e. a comparable
concentration to that of unlabeled bound FKBP in a 2.5 mg/ml TC
suspension. After incubation at 37 °C for 20 min, the TC vesicles
were separated from the soluble FKBP by centrifugation. Although the
distribution of total FKBP (as determined by
[
H]FK-816 binding) in the TC and soluble
fractions remained essentially the same before and after incubation,
the specific activity of bound [
S]FKBP increased
proportionally with a concomitant reduction in the specific activity of
soluble [
S]FKBP (not shown). Thus,
[
S]FKBP binding to TC reflects an exchange
phenomenon, in which binding is accompanied by displacement of
unlabeled, bound FKBP into the soluble fraction.
Figure 4:
Calcium and magnesium dependence of
calcium loading by control versus drug-treated terminal
cisternae. The net ATP-stimulated calcium loading rate was determined
for control TC (), drug-treated TC (
), and either control
or drug-treated TC in the presence of 5 µM ruthenium red
(
). A, calcium concentration dependence of the net
loading rate at a concentration of 3 mM MgCl
. B, magnesium concentration dependence of the net loading rate
initiated by addition of 50 µM CaCl
to the
cuvette. The results shown in this figure are typical of three
different TC preparations.
Likewise, the magnesium sensitivity of the CRC in control versus drug-treated TC can be assessed by monitoring the net calcium
loading rate at graded doses of Mg in response to a
large activating pulse (50 µM) of Ca
(Fig. 4B). At 4 mM Mg
,
the net loading rate is slow due to Ca
-induced
activation of both control and drug-treated TC. Drug-treated TC require
a greater concentration of Mg
to block
Ca
-induced activation of the CRC. One interpretation
of these results is that FKBP plays a role in ``fine-tuning''
the sensitivity of the CRC to divalent cations, i.e. the CRC
in drug-treated TC have an enhanced sensitivity to activating
Ca
ions (Fig. 4A) and/or a decreased
sensitivity to blockage by Mg
(Fig. 4B).
Figure 5:
Correlation of calcium loading rate with
dissociation of [S]FKBP-12. Prelabeled TC (96
pmol of [
S]FKBP-12 per mg of TC) were incubated
in IHM buffer containing 0 to 12.5 µM FK-590 (A)
or FK-977 (B) for 20 min at 37 °C. The amount of
[
S]FKBP-12 remaining in the pellet (
) was
measured and correlated with the net calcium loading rate (
)
determined in loading medium containing 10 mM MgCl
following the addition of 50 µM Ca
to the cuvette. The curves were generated by nonlinear regression
of the data as described under ``Experimental Procedures.''
The prelabeled TC in this experiment had a net calcium loading rate of
0.85 µmol of calcium per min per mg of TC. The EC
value for FK-590 to dissociate [
S]FKBP-12
and reduce the net loading rate (0.25 to 0.66 µM) was in
the same range for three preparations, while the range of EC
values for FK-977 (4.3 to 9.1 µM), which has a lower
affinity for FKBP-12 than FK-590, was consistently at least 10 times
greater for the same three preparations of
TC.
Figure 6:
Restoration of net calcium loading rate
with FKBP mutants. Typical traces for the calcium loading rate assay
are shown for untreated TC, drug-control TC, drug-treated TC, and
several different samples of reconstituted TC. The net calcium loading
rate was determined in loading medium containing 3 mM MgCl in response to multiple pulses of 12.5 µM CaCl
. The rates expressed for each treatment are the
average values obtained for only the first three pulses of calcium, as
detailed under ``Experimental Procedures.'' Table 4summarizes data obtained from a total of five different TC
preparations. The values in parentheses for the reconstituted TC are
PPIase activity of each FKBP mutant relative to wild-type (see Table 1).
We also
used the ``reconstitution methodology'' to determine whether
the PPIase-deficient mutants could restore the net calcium loading rate
of drug-treated TC. Fig. 6shows typical calcium loading rate
assays for seven different TC samples. The results from five different
experiments are summarized in Table 4. After all of the loading
assays were completed, a post-labeling assay was performed to estimate
the number of unoccupied binding sites remaining in each sample that
can be rapidly filled in the cold by direct binding of
[S]FKBP to unoccupied FKBP binding sites (see
the legend to Table 4). The net Ca
loading rate
is significantly reduced (to 63% of control TC) in drug-treated TC. The
drug-treated sample binds 85 pmol/mg of [
S]FKBP
as determined by the postlabeling protocol which indicates that most of
the bound FKBP had been dissociated from the CRC with drug. By
contrast, drug control TC have a similar Ca
loading
rate and bind a similar amount of [
S]FKBP as
untreated TC (21 versus 16 pmol/mg) in the postlabeling
protocol TC (Table 4).
Reconstitution of the drug-treated TC
was achieved as described under ``Experimental Procedures''
by adding back a 2-fold molar excess (over the concentration of FK-590)
of either wild type FKBP or the indicated PPIase-deficient FKBP mutants
to ice-cold samples of drug-treated TC. Reconstitution is confirmed by
the low level of [S]FKBP binding (15 to 21
pmol/mg) in the postlabeling protocol (Table 4). Reconstitution
of drug-treated TC with either wild-type FKBP or any one of the
different PPIase-deficient mutants (W59H, F36Y, or F99Y) significantly
enhances the net calcium loading rate to values which are, again,
similar to control TC (Fig. 4).
Thus, our studies confirm that FKBP modulates the function of the CRC, but suggests that this modulation is independent of PPIase activity of FKBP.
The finding that FKBP is bound to the ryanodine receptor
provides a system to study the possible role of this immunophilin in
cell function (Jayaraman et al., 1992). For this purpose, we
developed methodology to release tightly bound FKBP from the ryanodine
receptor of skeletal muscle TC. Such FKBP-stripped TC have altered
sensitivity, to Ca (enhanced) and Mg
(decreased) (Fig. 5, this study) and caffeine (enhanced),
and the channel becomes activated (enhanced open probability and longer
mean open times). These characteristics were restored to normal by
rebinding FKBP, which helps maintain the channel in a closed state.
Thus, some of the characteristics of the CRC are conferred in part by
FKBP (Mayrleitner et al.(1994), Timerman et al. (1993), Brillantes et al.(1994), and this study). The key
diagnostic features, such as interaction with specific ligands like
ryanodine and ruthenium red and the unitary conductance of the channel,
are intrinsic to the associated ryanodine receptor protomers, and their
cooperative association is modulated by FKBP (Mayrleitner et
al., 1994; Timerman et al., 1993; Brillantes et
al., 1994). These studies demonstrated that FKBP serves a
functional role by modulating the channel.
New to this study is the
finding that receptor-bound FKBP can be exchanged with added soluble
FKBP. This can be achieved with either wild-type or FKBP mutants with
altered FK-506 binding and PPIase activities. Thus, two methods were
available in our studies to assess the role of FKBP mutants on the
function of the ryanodine receptor, i.e. the dissociation and
reconstitution approach, and the newly developed exchange methodology.
Dissociation of FKBP enhances the open probability of the CRC which may
reflect an altered sensitivity of the channel to divalent cations, such
as Ca and/or Mg
(Fig. 4).
Rebinding recombinant FKBP, regardless of the PPIase activity, restores
the properties of the depleted channel back to control ( Fig. 6and Table 4). Exchange, with any of the FKBP mutants
including those devoid of PPIase activity, was found to regulate the
CRC similar to that of wild type (Table 4). Our studies indicate
that the PPIase activity of FKBP, as measured using peptide substrates,
is not critical for modulating the channel behavior of the CRC.
The
exchange methodology provides new insights into the association of FKBP
with the ryanodine receptor. In the absence of FKBP, the channel is
activated so that the net Ca loading rate of the
terminal cisternae is reduced due to the leak of Ca
through the channel (Fleischer et al., 1985). Rebinding
of FKBP restores the net Ca
loading to control
values. Exchange of bound FKBP with added soluble wild type or mutant
FKBP is time- and temperature-dependent. The exchange is much slower at
room temperature and very slow in the cold (perhaps a few percent in 2
h). The rate of the exchange process is dependent on the concentration
of FKBP in the medium (Fig. 2). The Scatchard plot gives a
single straight line indicative of one type of binding (exchange) site
with an apparent B
of
105 pmol/mg TC
protein or 129 pmol/mg when corrected for incomplete exchange in 20 min
(see Fig. 1). This value is essentially the same as that
obtained by drug binding isotherm (B
) which
measures the concentration of bound FKBP in terminal cisternae (124
pmol/mg TC). The apparent K
for exchange is 0.3
µM for wild-type FKBP. By contrast, FKBP is tightly bound
to TC. The ``off rate'' is negligible since most of the FKBP
binding sites (
94%) in isolated and multiple washed TC are filled
( Table 2and ``Results''). The dissociation of FKBP
with drug is likewise time- and temperature-dependent; about 80%
dissociation is obtained at 37 °C in 20 min (Timerman et
al., 1993). (
)In contrast to the relatively slow
exchange or dissociation of FKBP from the terminal cisternae at 37
°C, the ``on'' rate of FKBP to FKBP-stripped TC is rapid
(3 min or less) and takes place in the cold ( Table 2and Fig. 6).
In our previous studies, a key issue was whether the
effect of drugs on TC was due to dissociating FKBP from the ryanodine
receptor or due to the direct action of drug on the receptor itself. In
the studies presented here, new insight is provided. In the cold, (drug
control TC), the drug has essentially no effect on the characteristics
of the receptor ( Fig. 6and Table 4), whereas the same
sample incubated at 37 °C for 20 min (drug-treated TC) has altered
characteristics due to the release of bound FKBP as the drugFKBP
complex. FKBP-stripped TC can be separated from the soluble
FKBP
drug complex by centrifugation. The reassociation of FKBP
with the CRC occurs rapidly in the cold, when FKBP is added in excess
over drug to either FKBP-stripped TC or drug-treated TC. Such
reconstituted TC once again become regulated by FKBP, comparable to
untreated TC.
The concentration of FKBP in the myoplasm was measured
to be 3 µM. We estimate that a significant percentage
(perhaps 15%) of the FKBP in the muscle fiber is associated with the
SR. This calculation depends on a number of estimates: 1) myoplasm
occupies about 25% of the volume of the skeletal muscle fiber, 2) the
TC membrane occupies
1% of the fiber volume. This value is based
on morphometric measurements of the SR volume (Eisenberg, 1983) and
estimating that TC accounts for half of the SR, and one-third of the
volume of TC is occupied by membrane; 3) 1 ml of TC membrane contains
about 12 nmol of FKBP. This is based on the known content of FKBP in TC
(
100 pmol/mg of protein) and assuming that 1 ml of TC membrane
contains 120 mg of protein.
The concentration of FKBP in the
myoplasm is approximately 10-fold greater than the apparent K for exchange of soluble with bound FKBP (see
``Results'' and Table 1). Thus, at conditions of body
temperature and myoplasmic concentration of FKBP
, we
would expect rapid exchange of myoplasmic FKBP with that bound to the
ryanodine receptor in skeletal muscle. If this cycling were
regulatable, the cell would have a potential mechanism for
``fine-tuning'' the calcium sensitivity of the CRC.
Potentially, such a mechanism could be important in E-C coupling and in
cell signaling.
Our findings provide important leads to the study of the role of FKBP in E-C coupling. A variety of questions are relevant. What is the physiological significance of the putative exchange phenomenon of FKBP in the myoplasm with FKBP bound to the ryanodine receptor? Is there a mechanism to modulate the channel by changing the number of occupied FKBP binding sites on the CRC? If so, would such modulation be important in regulating E-C coupling?
Perhaps relevant
to the existence of a putative FK metabolite in the cell, Mack et
al.(1994) found that a family of natural products isolated from
the marine sponge (Ianthella basta), referred to as bastadins,
modulate the behavior of the skeletal muscle ryanodine receptor.
Bastadins have structural similarity to FK-506, and members of these
compounds have varied effects. For example, bastadin 5 significantly
enhanced high affinity ryanodine binding, increased dwell time of the
CRC, and decreased inhibition by Mg (Mack et
al., 1994). Dissociation of FKBP from the ryanodine receptor with
FK-506 antagonizes the unique action of bastadin 5, which does not
dissociate FKBP from the CRC. Is there a natural congener of FK-506 or
the bastadins in muscle that can modulate the association of the
FKBP/ryanodine with receptor interaction during E-C coupling?
Recent
studies indicate that immunophilins can have multiple subcellular
localizations in addition to the cytosol (Jayaraman et al.,
1992; Kunz and Hall, 1993) and thereby may be involved in regulating a
number of diverse cell processes. Of special note is a recent report
that FKBP is associated with type 1 transforming growth factor-
receptor (Wang et al., 1994). As with the ryanodine receptor,
FK-506 dissociates the interaction of FKBP-12 with the transforming
growth factor-
receptor.
It must be borne in mind that the PPlase assay makes use of a synthetic substrate which may not be an accurate measurement of activity toward the ryanodine receptor. Within this context, this study for the first time, directly addresses the role of PPIase activity in regulating channel function. We find that several different PPIase-deficient mutants bind to and regulate the CRC in a manner which is essentially identical with wild-type FKBP. Thus, our results suggest that the modulation of the CRC by FKBP is independent of PPIase activity.