©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of an Exchange Reaction between Soluble FKBP-12 and the FKBPRyanodine Receptor Complex
MODULATION BY FKBP MUTANTS DEFICIENT IN PEPTIDYL-PROLYL ISOMERASE ACTIVITY (*)

(Received for publication, August 26, 1994; and in revised form, November 3, 1994)

Anthony P. Timerman (§) Gregory Wiederrecht (1) Alice Marcy (2) Sidney Fleischer (¶)

From the  (1)Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235 and the Departments of Immunology and (2)Biophysical Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065-0900

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 FKBPbulletRyR 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 FKBPbulletRyR 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.


INTRODUCTION

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) (^1)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 FKBPbulletRyR 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)(4)/(RyR protomer)(4) (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 FKBPbulletFK-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.


EXPERIMENTAL PROCEDURES

Materials

The following materials were provided by Merck Research Laboratories: L-683,590, or simply FK-590 (a closely related structural analogue of FK-506; FK-590 is also referred to as FK-520 in the literature); L688-977 or FK-977, an analogue of FK-590 with weaker binding affinity for FKBP-12 than FK-590; L685-818 or FK-818, an analogue of FK-590 with similar binding affinity, yet the complex of FKBP with FK-818 does not bind to or inhibit calcineurin; [^3H]FK-816, a dihydropropyl derivative of FK-506; [S]Cys-labeled wild-type FKBP; and unlabeled wild-type and mutant FKBPs. FKBPs were expressed in Escherichia coli and purified in TSK column buffer (20 mM NaPO(4), pH 6.8, 50 mM Na(2)SO(4), 5 mM beta-mercaptoethanol, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride) by high performance liquid chromatography gel filtration chromatography on either: 1) a Bio-Sil TSK-125 column (21.5 mm times 60 cm, Bio-Rad) or 2) a TSK-GEL G2000SW column (21.5 mm times 60 cm, TosoHaas) as described previously (Wiederrecht et al., 1992). The gel exclusion purified proteins were concentrated by ultrafiltration in an Amicon stir cell fitted with a YM3 membrane. The specific activity of the [S]FKBP-12 used in the time span of these experiments ranged from 950 to 250 cpm/pmol.

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(4), 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.

General Methods

The protein concentrations of SR membrane fractions and purified FKBPs were determined by the Folin reaction (Lowry et al., 1951) using bovine serum albumin as standard. Unless indicated otherwise, all experiments were performed on at least three different preparations of TC vesicles with either wild-type or mutant FKBPs.

Isolation of Longitudinal Tubules, Terminal Cisternae, and Skeletal Muscle Cytosol

Longitudinal tubules (LT) and TC of SR were isolated from New Zealand White rabbit skeletal muscle as described previously (Chu et al., 1988; Saito et al., 1984; Inui et al., 1987). The cytosolic fraction of skeletal muscle was the high speed supernatant fraction obtained following sedimentation of the microsomes from the initial blendate or I-series (Chu et al., 1988).

[^3H]FK-816 Binding Assays

[^3H]FK-816 binding isotherms to the cytosol fraction of rabbit skeletal muscle was determined as described previously (Timerman et al., 1993) in the presence of 0.5% CHAPS from 1 to 30 nM [^3H]FK-816 (which has essentially the same binding characteristics as FK-506) using the Sephadex LH-20 column method (Handschumaker et al., 1984) to separate bound from free ligand.

Measurement of FKBP in the Myoplasm of Skeletal Muscle

The [^3H]FK-816 binding parameters to rabbit skeletal cytosol were determined by Scatchard analysis using conditions of the binding assay described above. The B(max) value for the cytosol fraction was obtained by multiplying the B(max) value for the I-series supernatant (Chu et al., 1988) by a factor of 20 (times5 (5:1 ratio of homogenization medium to muscle wet weight) and times4 (estimate that the cytosol accounts for about 25% of the tissue wet weight)).

FK-506 Binding Activity

This refers to the [^3H]FK-816 binding capacity for each wild type and mutant FKBP preparation. The binding capacity was determined from [^3H]FK-816 binding assays at a constant concentration of 25 nM or 1.86 ng/ml [^3H]FK-816 (which is 50 times the K(d) value of 0.5 nM for [^3H]FK-816 binding to wild-type FKBP-12) at a concentration range of 0.63 to 6.3 nM (7.4 to 74 ng of FKBP/ml).

Peptidyl-Prolyl cis/trans-Isomerase Activity of Recombinant FKBPs

The PPIase activity of each FKBP preparation is presented as the first order rate constant for the hydrolysis of N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide by chymotrypsin (Siekierka et al., 1989). The precision of the assay is ±0.1%.

Exchange Isotherms of [S]FKBP to Terminal Cisternae and Longitudinal Tubules

Exchange isotherms of [S]FKBP to TC or LT were performed by incubation of vesicles (at 2.5 mg/ml) with 0.125 to 3.0 µM [S]FKBP for 20 min at 37 °C. Nonspecific binding was estimated in the presence of a 10-fold excess of cold FKBP at each concentration of labeled protein. The nonspecific exchange is about 10% of specific [S]FKBP binding to TC, detectable after diluting the specific activity of [S]FKBP by 10-fold (Fig. 2). A more accurate estimate of nonspecific binding to TC requires significantly greater concentrations of unlabeled FKBP, or the data could be corrected by calculation. The data given in the tables were not corrected. Thus, the apparent B(max) value for specific (i.e. total minus nonspecific) binding of [S]FKBP to TC shown in Fig. 2B is an underestimate of the true B(max) value by about 10% or 10 pmol/mg of TC.


Figure 2: Concentration dependence for exchange of soluble [S]FKBP-12 with bound FKBP-12. A, exchange (binding) was performed by incubation of TC (bullet, circle) or LT (, down triangle) 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 (bullet, ) and nonspecific binding (circle, down triangle), 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(max) (``apparent'' B(max)) = 95.6 ± 28 pmol/mg of TC; and r (correlation coefficient) = 0.905 ± 0.007 (n = 4).



Preparation of Prelabeled Terminal Cisternae^2

One to ten ml of functionally competent TC vesicles were labeled by ``exchange'' of bound FKBP for soluble [S]FKBP as follows: 1) TC (at 2.5 mg/ml) were incubated with 2 µM [S]FKBP at 37 °C for 20 min; 2) free [S]FKBP was separated from bound [S]FKBP by sedimentation of TC at 30,000 times g(max) at 4 °C for either 15 min in a Beckman TL100.2 rotor or 30 min in a Beckman Ti70.1 rotor; 3) the pellet (designated ``prelabeled TC^2'') containing bound [S]FKBP was resuspended without a subsequent wash step to a final concentration of 2.5 mg/ml in IHM buffer containing 5% TSK column buffer, frozen in liquid nitrogen, and stored at -80 °C. These protocols generally yield 80% recovery of total protein and greater than 90% recovery of the net calcium loading rate (expressed as micromoles of calcium per min per mg of protein). The prelabeled TC is useful for quantitation of displacement (by exchange or dissociation) of bound [S]FKBP.

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).

Preparation of Drug-treated and FKBP-stripped Terminal Cisternae^2

The endogenous FKBP was dissociated from TC (2.5 mg/ml, which contains 0.25 µM endogenous FKBP) by incubation with either 1.25 or 2.5 µM FK-590 at 37 °C for 20 min. We refer to TC resulting from this treatment as ``drug-treated TC''; subsequently, ``FKBP-stripped TC'' can be obtained by sedimentation of drug-treated TC, in a Beckman TL100.2 rotor at 30,000 rpm for 15 min at 2 °C. The soluble FKBPbulletFK-590 complex remaining in the supernatant was discarded, while the pellet (designated FKBP-stripped TC) was resuspended to 2.5 mg/ml in IHM buffer containing 5% (v/v) TSK column buffer. Approximately 80 to 90% of the FKBP is removed from FKBP-stripped TC prepared in this manner. We have previously referred to this preparation as FKBP-deficient TC (Timerman et al., 1993).

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.

Preparation of Reconstituted TC^2

The binding of [S]FKBP to either drug-treated or FKBP-stripped TC vesicles is rapid (less than 3 min) even at 0-4 °C. This is in contrast to the time and temperature dependence for either drug-induced dissociation or the exchange of bound FKBP with soluble [S]FKBP (see ``Results''). Therefore, ice-cold samples (200 µl) of drug-treated TC were reconstituted with FKBP by simply adding back a 2-fold molar excess of recombinant FKBP (2.5 or 5.0 µM FKBP) over the concentration of FK-590. Samples of untreated TC, drug-treated, and reconstituted TC were stored on ice until the net calcium loading rate assay (see below) for each sample was completed (4-5 h). Afterwards, reconstitution of drug-treated TC by mutant FKBPs was routinely assessed by ``postlabeling'' with [S]FKBP as follows: 1) [S]FKBP (10 µl) was directly added to 50 µl of ice-cold samples (125 µg) of either control TC, drug-treated TC, or reconstituted TC to a final concentration of 2.5 µM [S]FKBP; 2) 25 µl of the mixture (50 µg) was immediately diluted into 200 µl of ice-cold IHM buffer, and the amount of [S]FKBP bound to the pellet, after sedimentation in a Beckman TL100.1 rotor, was determined as described above. Since the exchange reaction is negligible in this short time at 0-4 °C, the only significant binding to TC that occurs under these conditions is by direct binding (addition) of [S]FKBP to unoccupied FKBP binding sites on TC. Therefore, drug-treated TC (i.e. FKBP dissociated but not separated from TC with FK-590) bind significantly more [S]FKBP (85 pmol/mg) than do either control or reconstituted TC (about 15 pmol/mg TC) (Table 4)



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 FKBPbulletdrug 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 Rate Assays

The calcium loading rate of TC vesicles was monitored spectrophotometrically with the calcium indicator antipyrylazo III essentially as described previously (Fleischer et al., 1985; Timerman et al., 1993). TC vesicles (25 µg of protein) were added to 1 ml of loading medium (100 mM KPO(4), pH 7.0, 0.2 mM antipyrylazo III, and 1 mM ATP) containing 3 to 20 mM MgCl(2), as indicated. Ca loading was initiated with the addition of 1 mM ATP. Following the uptake of contaminating calcium, aliquots of 5 to 50 µM CaCl(2) (as indicated in each figure) were pulsed into the cuvette, and the calcium uptake rate was monitored by dual wavelength spectrophotometry (710-790 nm) in a Hewlett Packard 8451A diode array spectrophotometer. Following the various treatments described above (generally a 20-min incubation at 37 °C), TC samples were stored on ice until the calcium loading rate was measured with little, if any, loss of activity for up to 6 or 7 h.

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)^c] 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 (circle), F36Y (bullet), L30A (down triangle), W59H (), F99Y (), and Y26F (box). The EC values obtained from two experiments for each FKBP preparation is summarized in Table 1.




RESULTS

Exchange of Bound FKBP with Soluble [S]FKBP

We have previously developed methodology to release the tightly bound FKBP from TC by incubation with FK-590 or rapamycin. The drugs bind to bound FKBP in a temperature-dependent manner and the resulting drugbulletFKBP complex is released from the TC (Timerman et al., 1993). We now describe an exchange reaction in which bound FKBP is displaced from the FKBPbulletRyR complex with soluble FKBP added to a suspension of TC vesicles.

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 [^3H]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 (bullet), ambient temperature (22 °C, circle), 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(d)) of 0.33 ± 0.16 µM [S]FKBP (see Fig. 2legend). The apparent B(max) 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(max) of 129 pmol of [S]FKBP binding sites per mg of TC (95.6 pmol/mg times 1.35). The B(max) values for [^3H]FK-816 and [^3H]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(max) value for [S]FKBP binding (exchange) is essentially the same as the B(max) value for drug ([^3H]FK-816) binding in TC. Since there is only one high affinity [^3H]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(max) value (Fig. 2B) was essentially the same as that obtained by ligand binding ([^3H]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 [^3H]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.

FKBP Mutants Exchange for [S]FKBP from Prelabeled TC

Fig. 3shows the concentration dependence for exchange of either wild-type (hollow circles) or mutant FKBPs. The mutations are in highly conserved hydrophobic residues which affect drug binding and/or PPI ase activity. These mutants (Table 1), which vary widely in PPIase and FK-506 binding activity, effectively exchange for and displace the [S]FKBP from prelabeled TC. Comparison of the EC values for exchange with either the FK-506 binding or PPIase activity of each protein (Table 1) indicates that there is no apparent correlation with either FK-506 binding or PPIase activity. For example, the exchange EC for the three different PPIase-deficient mutants (Y26F, F99Y, and W59H) are only 2 to 8 times greater than the EC for wild-type FKBP (0.6 to 2.4 µMversus 0.3 µM, see Table 1), yet the EC for exchange by the Y26F mutant (which displays 20-30% of wild-type FK-506 binding and PPIase activity) was more than 15-fold higher than that of wild-type FKBP.

Concentration of FKBP in Skeletal Muscle Myoplasm

The concentration of myoplasmic FKBP was determined by both [^3H]FK-816 binding isotherms and semiquantitative Western blot analysis using sequence specific antisera raised against the amino or carboxyl terminus of FKBP and two-dimensional gel scanning densitometry versus a standard curve of 0.2 to 1.6 ng of recombinant FKBP (not shown). Both methods estimate the concentration of FKBP in skeletal muscle myoplasm to be about 3.0 ± 1.0 µM; the K(d) is 2.6 ± 0.1 nM. The use of the two procedures together indicates that the majority of [^3H]FK-816 binding equivalents in the cytosol is due to FKBP-12 rather than other FKBPs. Since the exchange reaction is rapid at 37 °C with an EC of 0.3 µM, these studies indicate that, in vivo, the bound FKBPbulletRyR complex exchanges with the myoplasmic pool of FKBP, which is 10 times higher than the EC for the exchange reaction.

Estimate of Unoccupied FKBP Binding Sites in Terminal Cisternae

The data compiled in Table 2summarizes three important observations regarding the temperature dependence for exchange, dissociation, and rebinding of FKBP to TC. First, as described above, the exchange reaction is enhanced with increasing temperatures up to 37 °C (Table 2, row 1 versus row 2). Second, [S]FKBP rapidly binds in the cold to drug-treated TC, i.e. TC in which the bound FKBP is first dissociated with FK-590 (Table 2, row 4). Therefore, FKBP rapidly binds to unoccupied binding sites in less than 3 min even at 0-4 °C. Finally, incubation of TC for up to several hours with FK-590 in the cold fails to dissociate FKBP from the TC as evidenced by the lack of [S]FKBP binding to drug-control TC (Table 2, row 3). With these points in mind, we estimate the concentration of unoccupied FKBP binding sites in TC to be 7 pmol/mg (see Table 2, row 2, 17.2-10.2). Since the concentration of FKBP binding sites in TC is approximately 125 pmol/mg, only about 6% (i.e. 7 out of 125 pmol/mg TC) of the total FKBP binding sites in extensively washed TC preparations are unoccupied. This observation explains why the zero time point at 0-4 °C shown in Fig. 1is as high as 15-20 pmol/mg TC, yet the total binding at 0-4 °C does not increase significantly thereafter.



Drug-treated TC Have an Altered Ca and/or Mg Sensitivity

Planar lipid bilayer experiments show that the CRC from FKBP-stripped TC, in comparison with channels from untreated TC, has a greater sensitivity to Ca for activation (Mayrleitner et al., 1994). Spectrophotometric assays of the net calcium loading rate of untreated versus drug-treated TC in response to graded doses of calcium (Fig. 4A) support our observation that dissociation of FKBP alters the calcium activation threshold of the CRC. The net calcium loading rate of TC is a function of both the energized uptake rate of Ca by the Ca pump minus the leak rate of calcium from the TC, specifically the Ca leak via the CRC. Thus, drugs which activate the channel (such as ryanodine) reduce the net loading rate, while drugs which block the channel (such as ruthenium red) enhance the net calcium loading rate of TC (Fleischer et al., 1985). In loading medium containing 3 mM MgCl(2), the net loading rate of both control and drug-treated TC is reduced with increasing concentrations of Ca (Fig. 4A). We interpret these results to reflect calcium-induced (calcium release) activation of both control and drug-treated TC. At concentrations of 5-20 µM Ca, there is a large difference (about 50%) between the net loading rates of control versus drug-treated TC, which reflects the enhanced calcium sensitivity of the FKBP-deficient CRC. The loading rates in the presence of 5 µM ruthenium red which block the CRC (Fig. 4A) are the same for both control and FKBP-stripped channels (Timerman et al., 1993). The enhanced Ca sensitivity of FKBP-stripped TC (Timerman et al., 1993), using this spectrophotometric assay, provides a simple and reliable assay for the functional association of FKBP with the CRC. Longitudinal tubules of SR, which are devoid of RyR, do not display calcium-induced calcium release or respond to treatment with immunosuppressive drug (up to 10 µM) (not shown and Timerman et al.(1993)).


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 (circle), and either control or drug-treated TC in the presence of 5 µM ruthenium red (bullet). A, calcium concentration dependence of the net loading rate at a concentration of 3 mM MgCl(2). B, magnesium concentration dependence of the net loading rate initiated by addition of 50 µM CaCl(2) 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).

Correlation of the Net Calcium Loading Rate with Dissociation of Bound FKBP-12

Our previous reconstitution studies demonstrated that FKBP modulates the activity of the CRC. A complementary experiment is provided in Fig. 5, where the differential effectiveness of FK-590 versus FK-977 to dissociate bound [S]FKBP from prelabeled TC is compared. FK-590 is 10-20 times more effective in dissociating bound [S]FKBP than FK-977 (Fig. 5, A versus B), reflecting the greater affinity for FKBP for FK-590 over FK-977. Dissociation of [S]FKBP is also observed with FK-977 but requires higher concentrations of drug (Fig. 5B). The release of bound [S]FKBP from prelabeled TC by each drug parallels the reduced net calcium loading rate for both drugs. FK-818, a ligand which has an affinity similar to FK-590, has a similar EC to both dissociate [S]FKBP and reduce the net calcium loading rate (not shown). These studies show that: 1) there is a direct correlation between the affinity of each drug for FKBP with the concentration required to dissociate [S]FKBP; 2) there is a concomitant reduction in the net calcium loading rate with the release of bound FKBP; 3) FK-590 reduces the calcium loading rate only when TC are incubated with the drug under conditions (higher temperatures) which dissociate bound FKBP. Thus, the calcium loading rate of drug-control TC is similar to untreated TC ( Fig. 6and Table 4). Taken together, these experiments ( Fig. 5and Fig. 6and Table 4) confirm that the activity of the CRC is directly modulated by its association with FKBP.


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 (bullet) was measured and correlated with the net calcium loading rate (down triangle) determined in loading medium containing 10 mM MgCl(2) 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(2) in response to multiple pulses of 12.5 µM CaCl(2). 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).



Modulation of the Ryanodine Receptor by PPIase-deficient FKBP Mutants

The role of PPIase activity of FKBP on the function of the CRC was assessed by measuring the net Ca loading rate of TC vesicles in which the bound wild-type FKBP was replaced with FKBPs that are practically devoid of PPIase activity, such as W59H (0.3% activity), F36Y (5% activity), or F99Y (0 ± 0.1% activity). The first protocol utilized the ``exchange methodology.'' Prelabeled TC were used to correlate displacement of [S]FKBP following re-exchange with unlabeled FKBP (Table 3). As positive control, dissociation of more than 80% of the [S]FKBP from prelabeled TC with FK-590 (drug-treated TC) reduced the net calcium loading rate to 54% of control (Table 3, row 2). By contrast, the calcium loading rate of prelabeled TC is essentially unchanged when more than 80% of the [S]FKBP is displaced by exchange with unlabeled, wild type FKBP-12 (re-exchanged with wild type, row 3 of Table 3). The net calcium loading rate was also essentially unchanged with each of the PPIase-deficient FKBP-12 mutants, i.e. W59H, F36Y, or F99Y (Table 3, rows 4-6). The apparent reduction in the net loading rate of 15-20% following re-exchange with W59H appears to be a nonspecific effect since a similar reduction is observed when W59H is incubated with prelabeled TC in the cold in which exchange does not occur (not shown). Only the W59H mutant displayed such a nonspecific effect on the net loading rate.



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.


DISCUSSION

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(max) 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(max)) which measures the concentration of bound FKBP in terminal cisternae (124 pmol/mg TC). The apparent K(d) 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). (^3)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 drugbulletFKBP complex. FKBP-stripped TC can be separated from the soluble FKBPbulletdrug 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(d) 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-beta receptor (Wang et al., 1994). As with the ryanodine receptor, FK-506 dissociates the interaction of FKBP-12 with the transforming growth factor-beta 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.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL32711 and grants from the Muscular Dystrophy Association of America. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Dept. of Chemistry, University of Wisconsin, Stevens Point, WI 54481.

To whom correspondence should be addressed. Tel.: 615-322-2132; Fax: 615-343-6833.

(^1)
The abbreviations used are: RyR, ryanodine receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CRC, calcium release channel; E-C coupling, excitation-contraction coupling; FKBP refers to FKBP-12, the FK-506 binding protein of 12 kda; GST/FKBP is the fusion protein of glutathione transferase with FKBP; IHM, imidazole homogenization medium; LT, longitudinal tubules of sarcoplasmic reticulum; PPIase, cis/trans-peptidyl-prolyl isomerase; SR, sarcoplasmic reticulum; TC, terminal cisternae of sarcoplasmic reticulum.

(^2)
Summary of terms used for modified terminal cisternae samples: TC vesicles typically bind about 100 pmol of [^3H]FK-816 per mg of TC (Timerman et al., 1993). This is equivalent to a concentration of bound FKBP of about 0.25 µM at 2.5 mg of protein/ml of suspension of TC. A variety of treatments were carried out at this concentration of TC to yield modified types of TC, defined as follows. 1) Drug-control TC, samples incubated with FK-590 for up to several hours in the cold (0-4 °C). Little or no drug binding or dissociation of bound FKBP occurs under these conditions. 2) Drug-treated TC, bound FKBP is dissociated from the CRC by incubation at 37 °C for 20 min with 1.25 or 2.5 µM FK-590 (i.e. 5 to 10 times higher than the concentration of bound FKBP). 3) FKBP-stripped TC, drug-treated TC which have been sedimented to remove the released soluble FKBP which remains in the supernatant as the drug complex (80 to 90% of FKBP is removed). 4) Reconstituted TC, TC prepared from drug-treated or FKBP-stripped TC by rebinding FKBP in the cold (0-4 °C) using a 2-fold molar excess of FKBP to drug (1.25 or 2.5 µM) which was used to dissociate bound FKBP. 5) Exchanged TC, bound FKBP is exchanged with soluble FKBP (2 µM) for 20 min at 37 °C. 6) Prelabeled TC, TC exchanged with [S]FKBP. 7) Re-exchanged TC, prelabeled TC which have been exchanged yet again with unlabeled FKBP.

(^3)
A. P. Timerman and S. Fleischer, unpublished studies.


ACKNOWLEDGEMENTS

We thank Sebastian Barg of our laboratory for his comments on the manuscript.


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