©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ryanodine Receptor-Ankyrin Interaction Regulates Internal Ca Release in Mouse T-lymphoma Cells (*)

(Received for publication, January 13, 1995; and in revised form, May 24, 1995)

Lilly Y. W. Bourguignon (1)(§) Arthur Chu (1) H. Jin (1) Neil R. Brandt (2)

From the  (1)Department of Cell Biology and Anatomy and the (2)Department of Molecular Pharmacology, University of Miami Medical School, Miami, Florida 33101

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In this study, we have identified and partially characterized a mouse T-lymphoma ryanodine receptor on a unique type of internal vesicle which bands at the relatively light density of 1.07 g/ml. Analysis of the binding of [H]ryanodine to these internal vesicles reveals the presence of a single, low affinity binding site with a dissociation constant (K) of 200 nM. The second messenger, cyclic ADP-ribose, was found to increase the binding affinity of [H]ryanodine to its vesicle receptor at least 5-fold (K 40 nM). In addition, cADP-ribose appears to be a potent activator of internal Ca release in T-lymphoma cells and is capable of overriding ryanodine-mediated inhibition of internal Ca release.

Immunoblot analyses using a monoclonal mouse anti-ryanodine receptor antibody indicate that mouse T-lymphoma cells contain a 500-kDa polypeptide similar to the ryanodine receptor found in skeletal muscle, cardiac muscle, and brain tissues. Double immunofluorescence staining and laser confocal microscopic analysis show that the ryanodine receptor is preferentially accumulated underneath surface receptor-capped structures. T-lymphoma ryanodine receptor was isolated (with an apparent sedimentation coefficient of 30 S) by extraction of the light density vesicles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in 1 M NaCl followed by sucrose gradient centrifugation. Further analysis indicates that specific, high affinity binding occurs between ankyrin and this 30 S lymphoma ryanodine receptor (K = 0.075 nM). Most importantly, the binding of ankyrin to the light density vesicles significantly blocks ryanodine binding and ryanodine-mediated inhibition of internal Ca release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of ryanodine receptor-mediated internal Ca release during lymphocyte activation.


INTRODUCTION

Upon agonist stimulation, most cells release Ca sequestered in internal storage sites through Ca release channels, identified as the ryanodine receptor (RYR)()(1) or the inositol 1,4,5-trisphosphate (IP) receptor(2) . These two Ca channels are homotetrameric megaproteins (subunit molecular mass of 500 kDa for the ryanodine receptor and 270 kDa for the IP receptor) which share extensive homology in their C-terminal portion where the majority of the transmembrane segments are predicted to be located(2) .

Ryanodine receptors were originally found in the sarcoplasmic reticulum of skeletal muscle (type 1 receptor, RYR1) and cardiac muscle (type 2 receptor, RYR2)(3, 4) . The two forms of the ryanodine receptors appear to be encoded by different genes(3, 4) . Ca release from the sarcoplasmic reticulum through these receptors plays a central role in regulating the contraction of skeletal and cardiac muscle fibers. In vivo, the Ca release channel in skeletal muscle is activated by direct physical coupling to the voltage sensor in the transverse tubule (5, 6) while the cardiac Ca channel is activated by Ca influx through plasma membrane-associated Ca channels(7, 8) . Recently, a third ryanodine receptor gene (type 3 receptor, RYR3) has been detected in brain tissues(9) . Since this new isoform of RYR is also found in smooth muscle tissue and several types of non-muscle cells(4, 9, 10) , the concept that ryanodine receptors are muscle-specific Ca channels must be changed. Furthermore, it has been suggested that activation of ryanodine receptors by a second messenger (e.g. cyclic ADP-ribose (cADPR)) plays a critical role in intracellular Ca signaling occurring during agonist-induced cell activation in non-muscle cells(11, 12) .

Non-muscle cells, such as mouse T-lymphoma cells, may possess a specialized sarcoplasmic reticulum-like organelle required for regulating internal Ca release. It has been shown that IP is involved in stimulating ``light density vesicles'' in mouse T-lymphoma cells to release Ca(13) . Recent cloning and cDNA sequence analysis show that the brain type of ryanodine receptor (RYR3) transcript is expressed in human Jurkat T-lymphocyte cells(14) . However, direct biochemical evidence for the existence of a non-muscle ryanodine receptor (with a separate type of intracellular Ca-release channel) in lymphoid cells has not been reported.

In this paper, we have isolated and partially characterized a mouse T-lymphoma ryanodine receptor from a type of internal vesicle which bands at the relatively light density of 1.07 g/ml. Immunoblot analyses using a monoclonal mouse anti-ryanodine receptor antibody indicate that mouse T-lymphoma cells contain a 500-kDa polypeptide similar to the ryanodine receptor found in skeletal muscle, cardiac muscle, and brain tissues. Most importantly, we have determined that ankyrin (a membrane-associated cytoskeletal protein) binds specifically to the mouse T-lymphoma ryanodine receptor, and that this binding significantly inhibits ryanodine binding to ryanodine receptor and blocks ryanodine-mediated inhibition of internal Ca release. These results strongly suggest that the interaction between ryanodine receptor and ankyrin may play an important role in the regulation of internal Ca release during lymphocyte activation.


MATERIALS AND METHODS

Reagents

[H]Ryanodine (specific activity 54.7 Ci/mmol) was purchased from DuPont NEN. CaCl (specific activity 5-30 Ci/g) was obtained from ICN. cADPR was purchased from Amersham.

Cell Culture

The mouse T-lymphoma BW 5147 cell line (an AKR/J lymphoma line) was grown at 37 °C in 5% CO, 95% air using Dulbecco's modified Eagle's medium supplemented with 10% heatinactivated horse serum (Life Technologies, Inc.), 1% penicillin, and 1% streptomycin.

Cellular Fractionation

The cells (suspended in 50 ml of ice-cold buffer consisting of 15 mM KCl, 1.5 mM Mg(OAc), 1 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM HEPES (pH 7.0)) were disrupted by nitrogen cavitation in an Artisan homogenizer (Artisan Industries, Inc., Waltham, MA) held at 0 °C using a pressure of 600 p.s.i. for 15 min. After disruption, one-tenth volume of 700 mM KCl, 40 mM Mg(OAc), 1 mM dithiothreitol, 400 mM HEPES (pH 7.0), 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF was added, and nuclei were removed by centrifugation at 500 g for 4 min. The resulting supernatant was layered on a discontinuous sucrose gradient consisting of 0%, 15%, 25%, 35%, 40%, 50% sucrose (w/w) in a buffer containing 10 mM HEPES (pH 7.0), 50 mM KCl, 1 mM dithiothreitol, 2 mM MgCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF. The gradient was centrifuged in a Beckman SW28 rotor at 25,000 rpm for 16 h as described previously(13) . The membranous materials located in various sucrose layers were collected for further biochemical analyses including enzyme marker assays, ryanodine binding assays, Ca flux measurement, and immunoblotting techniques as described below.

Enzyme Marker Assays

Na/K-ATPase activity was used as a specific enzyme marker for plasma membrane(15, 16) . NADPH-dependent cytochrome c reductase and Sulfatase C activities were used as independent markers of endoplasmic reticulum(17, 18) . Galactosyltransferase activity, which was used as a Golgi marker, was assayed according to the procedures described previously(19) . -N-Acylglucosaminidase was used as a lysosomal marker(20, 21) . Protein concentrations were determined using the Bio-Rad Protein Assay Kit.

Double Immunofluorescence Staining

Mouse T-lymphoma cells were washed with Dulbecco's modified Eagle's medium and resuspended in the same medium. Fluorescein-conjugated concanavalin A (FITC-Con A) (20 µg/ml) was added directly to the cell suspension at 0 °C for 30 min. Cells were washed with Dulbecco's modified Eagle's medium three times followed by incubation at 37 °C for 15 min to induce patched or capped structures. Cells were then fixed in 2% paraformaldehyde containing 0.1 M phosphate buffer (pH 7.2). In some cases, cells were fixed in 2% paraformaldehyde without pretreatment with fluorescein isothiocyanate-ConA. Subsequently, fixed cells were either rendered permeable by 90% ethanol or without any ethanol permeabilization followed by staining with an affinity-purified monoclonal mouse anti-ryanodine receptor antibody (RYR.1, it was raised against the C terminus cytoplasmic domain of the ryanodine receptor). These samples were then labeled with rhodamine-conjugated goat anti-mouse IgG. To detect nonspecific antibody binding, cells were incubated with normal mouse serum followed by rhodamine-conjugated goat anti-mouse IgG. No staining was observed in such control samples. Fluorescence-labeled samples were examined with a confocal laser scanning microscope (MultiProbe 2001 Inverted CLSM system, Molecular Dynamics).

SDS-PAGE and Autoradiographic Analyses

Electrophoresis was conducted using a 5% polyacrylamide SDS-PAGE slab gel and the discontinuous buffer system described by Laemmli(22) . For autoradiographic analysis, all gels were vacuum-dried and exposed to Kodak x-ray film (X-Omat XAR-5).

Preparation of Ankyrin

Human erythrocyte ankyrin was purified by the procedure of Bennett and Stenbuck(23) .

Ryanodine Binding Assays

Lymphoma light density vesicles (isolated from the 15-25% sucrose interface according to the procedures described above) were incubated in a solution containing 20 mM Tris-HEPES (pH 7.2), 150 mM KCl, 1 mM CaCl, 0.2 mM PMSF, 1 mM iodoacetamide, 5 µg of aprotinin, 5 µg of leupeptin, and 10 nM [H]ryanodine in the presence of various concentrations of unlabeled ryanodine (ranging from 50 nM-100 µM) for 16 h at 4 °C. Total Ca concentration in the solution was monitored by atomic absorption spectroscopy using a Perkin-Elmer model 5000 spectrophotometer equipped with a Perkin-Elmer HGA500 graphite furnace. A computer program, taking into account pH, Ca, and K, was used to calculate the concentration of EGTA needed to adjust the free Ca concentration. In some cases, either cyclic ADP-ribose (cADPR) (1-3 µM) or cytoskeletal proteins (e.g. ankyrin (10 µg/ml) or fodrin (10 µg/ml)) was also included in the ryanodine binding solution. The binding reaction was terminated by adding a solution containing 150 mM NaCl, 20 mM Tris-HEPES (pH 7.2), 1 mM MgCl, and 1 mM EGTA and filtrating through a Millipore filter (HAWP, 0.45 µm). The filter-associated radioactivity was analyzed by liquid scintillation counting. The results were expressed as ``specific binding'' in which the nonspecific binding (defined as the radioactivity associated with samples and the filter in the presence of 10M unlabeled ryanodine) was subtracted from the total [H]ryanodine binding. The background represents approximately 20% of the total [H]ryanodine binding activity.

Isolation of 30 S Ryanodine Receptor

Membranes (e.g. mouse lymphoma light density vesicles, rabbit skeletal muscle sarcoplasmic reticulum, rat cardiac muscle sarcoplasmic reticulum, and rat brain membrane vesicles) were solubilized in a solution containing 1 M NaCl solution, 20 mM MOPS, 0.25% CHAPS/phosphatidylcholine (10:1), and protease inhibitors, a modified procedure from a previous report (24) . Solubilized proteins were centrifuged on 5-30% linear sucrose density gradients in a Beckman SW50 for 16 h at 100,000 g. Gradient fractions were analyzed by postlabeling with [H]ryanodine which involves an incubation of various gradient fractions in a solution containing 20 mM Tris-HEPES (pH 7.2), 150 mM KCl, 1 mM CaCl, 0.2 mM PMSF, 1 mM iodoacetamide, 5 µg of aprotinin, 5 µg of leupeptin, and 10 nM [H]ryanodine in the presence of various concentrations of unlabeled ryanodine (ranging from 50 nM-100 µM) for 16 h at 4 °C. To determine [H]ryanodine binding, the individual fraction was applied onto a small (1-ml) Sephadex G-50 column equilibrated in a solution containing 1 M NaCl, 20 mM MOPS, 0.25% CHAPS/phosphatidylcholine, and protease inhibitors. The Sephadex G-50 eluent (i.e. [H]ryanodine-bound material) was collected by low speed centrifugation (500 g), and the radioactivity was analyzed by liquid scintillation counting.

Ankyrin Binding Assay

Ankyrin was labeled with NaI using IODOBEADS. I-Ankyrin (50 ng) was incubated with a nitrocellulose sheet coated with purified ryanodine receptors (30 S molecule obtained from lymphoma light density vesicles, skeletal muscle sarcoplasmic reticulum, cardiac muscle sarcoplasmic reticulum, or brain membrane vesicles according to the procedures described above) in the presence of various concentrations of unlabeled ankyrin (ranging from 0.01 nM-1.00 µM) in a binding solution containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Triton X-100, and 0.1% bovine serum albumin for 16 h at 4 °C. Following incubation, the nitrocellulose sheets were washed 5 times with the same binding solution and counted in a counter. The results were expressed as specific binding in which the background level of binding was subtracted. As a control, we removed ryanodine receptors by incubating the 30 S peak fraction with anti-ryanodine receptor antibody-conjugated Sephadex beads. No specific ankyrin binding was detected in these ryanodine receptor-free samples.

CaFlux Measurement in Light Density Vesicles

Ca fluxes were studied in a reaction mixture containing 20 mM Tris-HEPES (pH 7.2), 150 mM KCl, 0.2 mM PMSF, 1 mM iodoacetamide, 5 µg of aprotinin and 5 µg of leupeptin, 1 mMCaCl in the presence or absence of various concentrations of ryanodine (10-100 µM) overnight. In some cases, either cyclic ADP-ribose (cADPR) (1-3 µM) or ankyrin (10 µg/ml) was also included in the reaction medium in which the free Ca was preadjusted to 0.1 µM. The Ca efflux was quenched by the addition of 5 mM LaCl. The amount of Ca released from the light density vesicles was determined by a filtration method using Millipore filters (HAWP, 0.45 µm) and washing with a buffer containing 120 mM KCl and 20 mM Tris-HEPES (pH 7.2). The filter-associated radioactivity was analyzed by liquid scintillation counting.

Immunoblotting Techniques

Protein samples including purified lymphoma 30 S molecule, lymphoma light density vesicles (collected from 15-25% sucrose interface), lymphoma total cellular proteins, rabbit skeletal muscle membranes, rat cardiac muscle membranes, and rat brain membrane vesicles were analyzed by a 5% polyacrylamide gel electrophoresis followed by transferring to nitrocellulose sheets. Ryanodine receptor-related immunoreactive proteins were detected by an enhanced chemiluminescence method using a 1:600 dilution of an affinity-purified monoclonal mouse anti-ryanodine receptor antibody (RYR.1) (1 mg/ml) and a 1:7000 dilution of horseradish peroxidase-conjugated sheep anti-rat IgG (1 mg/ml) (Amersham) followed by exposure to Kodak XAR-5 film. Normal mouse serum was used as a control.


RESULTS AND DISCUSSION

Subcellular Localization of the Ryanodine Receptor on CaStorage Vesicles in Mouse T-lymphoma Cells

It is well known that Ca plays an important role in the regulation of a large number of cellular activities including lymphocyte receptor capping(25) . It has been suggested that the increase in intracellular Ca concentration following the addition of ligand (or agonist) results from either Ca release from intracellular storage vesicles or exchange with extracellular Ca(2) . Previously, we have shown that an IP receptor-gated Ca channel is involved in the internal Ca release in mouse T-lymphoma cells(13) . A recent study reports that lymphocytes contain a brain-type ryanodine receptor transcript which appears to be involved in T-cell function(14) . However, the biochemical properties and regulatory aspects of this ryanodine receptor have not been determined.

In order to characterize the lymphocyte ryanodine receptor, we have carried out the following subcellular localization experiments. First, mouse T-lymphoma cells were homogenized by nitrogen cavitation, and vesicle populations were fractionated by discontinuous sucrose density gradient centrifugation (i.e. 0%, 15%, 25%, 35%, 40%, and 50% sucrose) (Table 1). Enzyme marker analyses (13) indicate that the major organelle membranes (e.g. Golgi membrane, endoplasmic reticulum, lysosomal membranes, and other large particles (e.g. membrane-bound ribosomes)) are located at the 25-35% sucrose interface (fraction C), the 35-45% sucrose interface (fraction D), and the 40-50% sucrose interface (fraction E) (Table 1). Mouse T-lymphoma ryanodine binding sites, however, are preferentially located in the light density vesicle fraction at the 15-25% sucrose interface-fraction B (Table 1).



In this paper we have focused on the ryanodine binding sites detected in the light density vesicles (fraction B with a density of approximately 1.07 g/ml) (Table 1). The fact that the mouse T-lymphoma IP receptor is also found in this light density vesicle fraction (13) indicates that either the two types of receptors are located in different vesicle populations which share similar isopycnic (density) points, or both the ryanodine receptor and the IP receptor are located on the same vesicles. The relationship between the ryanodine receptor and the IP receptor in mouse T-lymphoma cells is currently under investigation.

Ryanodine Binding Properties and Effects of Cyclic ADP-Ribose (cADPR) on the Ryanodine-sensitive CaChannel

The affinity of [H]ryanodine for the light density vesicles was measured in binding inhibition experiments using increasing concentrations of unlabeled ryanodine. The displacement curve reveals the presence of a single low affinity ryanodine binding site with a dissociation constant (K) of 200 ± 10 nM (Fig. 1). This K is similar to that for the low affinity sites found on skeletal muscle sarcoplasmic reticulum (4, 26, 27) and reported to be associated with inhibition of Ca channel activity(4, 28) . The high affinity ryanodine binding site (K 2-5 nM) locks ryanodine receptors into an open state upon binding of ryanodine in both skeletal (1, 4) and cardiac (1, 4) muscles. This high affinity ryanodine binding site was not detected in resting (unstimulated) mouse T-lymphoma cells.


Figure 1: Binding of [H]ryanodine to light density vesicles. Light density vesicle membranes (collected from 15-25% sucrose interface) were incubated with [H]ryanodine in the presence of various concentrations of unlabeled ryanodine in binding buffer as described under ``Materials and Methods.'' , without any treatment; , with 1 µM cADPR treatment. In Fig. 1, the results were expressed as specific binding. Specifically, the concentration of [H]ryanodine was held constant, and increasing amounts of unlabeled ryanodine (ranging from 10M to 10M) were added to adjust the total ryanodine concentration. Nonspecific binding was defined as the [H]ryanodine binding occurring in the presence of 10M unlabeled ryanodine. The amount of nonspecific binding did not exceed 20% of total [H]ryanodine binding at any unlabeled ryanodine concentration below 10M and was subtracted from the total [H]ryanodine binding at all ryanodine concentrations (10M-10M) to give the specific binding. The maximal level of [H]ryanodine binding to lymphoma light density vesicles is approximately 30 pmol/mg of protein. K was measured as 50% inhibition of specific [H]ryanodine binding. The numbers shown in this figure were the averages of triplicate determinants in three experiments, which varied by less than 5% (n = 9).



In nonexcitable cells, most extracellular signals activate Ca release from internal stores through the production of the second messenger, IP(2) . Recent studies on Ca mobilization, however, suggest that second messengers other than IP may be involved in the regulation of intracellular Ca concentration. A likely candidate for such as second messenger is cyclic ADP-ribose (cADPR), a metabolite of NAD, which has been shown to be a potent activator of internal Ca release in sea urchins (29, 30) and a modulator of the ryanodine-sensitive Ca release in both cardiac and brain microsomes(31) . Activation of ryanodine receptors by cADPR is modulated by calmodulin which serves as an inhibitor for internal Ca release(32) . In this study, we have found that cyclic ADP-ribose (cADPR) increases the binding affinity of [H]ryanodine to its receptor at least 5-fold (K 40 nM) (Fig. 1). It is possible that low affinity ryanodine binding sites are normally expressed in the resting (unstimulated) lymphoid cells. These low affinity ryanodine binding sites may then be converted into higher affinity ryanodine binding sites when a second messenger, such as cADPR, is formed during lymphocyte activation by agonists. It has been shown that both cADPR and its breakdown product, ADP-ribose, are capable of influencing the Ca release channel(33) . In this study we cannot rule out the possibility that cADPR and/or ADP-ribose are responsible for the increase of the K of [H]ryanodine binding.

We have also found that the light density vesicles display Ca release activities (Table 2) which can be selectively inhibited by ryanodine. These findings suggest that (i) the mouse T-lymphoma cells' light density vesicles contain a ryanodine-sensitive Ca channel (Tables I, II, and Fig. 6, panel 1), (ii) the cADPR is a potent activator of internal Ca release; and (iii) cADPR-mediated activation is not inhibited by ryanodine (Table 2). Presently, the mode of action of cADPR in regulating Ca channel activity is not fully established. It is speculated that cADPR displays stimulatory effects on both ryanodine-sensitive and ryanodine-insensitive Ca channel activities. Inhibition of cADPR-stimulated ryanodine-sensitive Cachannels by ryanodine may be overridden by the extensive cADPR stimulation on ryanodine-insensitive Cachannels under our assay conditions. This hypothesis is currently under investigation in our laboratory. It is noteworthy that a similar overriding of ryanodine inhibition of internal Ca release by cADPR has also been reported to occur in cardiac muscle sarcoplasmic reticulum and brain vesicles(31) .




Figure 6: Effect of ankyrin on Ca release in lymphoma light density vesicles. Panel 1A, untreated vesicles; panel 1B, ryanodine-treated vesicles; panel 2A, ankyrin-treated vesicles; panel 2B, ankyrin-treated vesicles followed by ryanodine treatment.



Some studies also indicate that cADPR does not act as a direct endogenous activator of Ca release activity associated with the skeletal or cardiac muscle ryanodine receptor(33, 34) . Structurally, the skeletal muscle ryanodine receptor (RYR1) and the cardiac ryanodine receptor (RYR2) are different from the brain and lymphoma ryanodine receptor (RYR3). It is possible that: (i) cADPR displays high affinity binding with RYR3 but has a low binding affinity with RYR1 and RYR2 or (ii) the binding affinity for cADPR to RYR3 may be higher than that in RYR1 and RYR1. The effective interaction between cADPR and RYR3 (but not RYR1/RYR2) may activate Ca release channels only in RYR3-containing tissues and cells. A systematic analysis of cADPR binding to the three types of ryanodine receptors (i.e. RYR1, RYR2, and RYR3) is needed to test this hypothesis.

Immunological and Structural Analyses of Mouse T-lymphoma Ryanodine Receptor

Extraction of the light density vesicles with CHAPS in 1 M NaCl, followed by sucrose gradient centrifugation and postlabeling with [H]ryanodine, yields a single peak with a sedimentation coefficient of approximately 30 S, similar to the skeletal muscle ryanodine receptor (Fig. 2A). SDS-PAGE analysis followed by silver staining of the 30 S peak material reveals a single polypeptide (molecular mass of 500 kDa) (Fig. 2B). Using immunoblotting techniques, we have found that the monoclonal anti-ryanodine receptor antibody (RYR.1) recognizes a single polypeptide (with a molecular mass of 500 kDa) present among all lymphoma cellular proteins (Fig. 3, E and F). This antibody also binds the ryanodine receptor (Fig. 3D) isolated from skeletal muscle (Fig. 3A), cardiac muscle (Fig. 3B), brain tissues (Fig. 3C), and the lymphoma 30 S peak fraction (Fig. 3D). These findings clearly indicate that mouse T-lymphoma cells do, in fact, contain ryanodine receptors.


Figure 2: Isolation of 30 S lymphoma ryanodine receptor. A, lymphoma light density vesicles were solubilized in a solution containing 1 M NaCl solution, 0.25% CHAPS/phosphatidylcholine, and protease inhibitors. Solubilized proteins were centrifuged on 5-30% linear sucrose density gradients in a Beckman SW50 for 16 h at 100,000 g. Gradient fractions were analyzed by postlabeling with [H]ryanodine as described under ``Materials and Methods.'' As a protein standard, skeletal muscle ryanodine receptor (with a sedimentation value of 30 S) was used to determine the s value of the [H]ryanodine binding peak. B, polypeptide pattern revealed by SDS-PAGE analysis followed by silver staining of the 30 S peak material.




Figure 3: Immunoblot analyses of ryanodine receptor. A, skeletal muscle membranes incubated with monoclonal anti-ryanodine receptor antibody (RYR.1) antibody. B, cardiac muscle membranes incubated with monoclonal anti-ryanodine receptor antibody (RYR.1) antibody. C, brain membranes incubated with monoclonal anti-ryanodine receptor antibody (RYR.1) antibody. D, lymphoma 30 S molecule incubated with monoclonal anti-ryanodine receptor antibody (RYR.1) antibody. (Similar results were observed when lymphoma light density vesicles were incubated with monoclonal anti-ryanodine receptor antibody (RYR.1) antibody (data not shown).) (As a control, normal mouse IgG was used. No staining was detected on these samples (data not shown).) E, lymphoma total cellular proteins were incubated with monoclonal anti-ryanodine receptor antibody (RYR.1) antibody. (As a control, normal mouse IgG was used. No staining was detected on these samples (data not shown).) F, Coomassie-stained lymphoma total cellular proteins. (The protein concentration used in these experiments was approximately 100 µg/gel lane.)



In Fig. 4, we have shown that if the cells were prefixed with 2% formaldehyde, without being permeabilized by ethanol treatment, no anti-ryanodine receptor-mediated staining can be detected (Fig. 4A). This finding suggests that ryanodine receptor is not located on the surface membrane of mouse T-lymphoma cells. When cells (in the absence of any external ligand challenge) were fixed with 2% formaldehyde followed by ethanol permeabilization and anti-ryanodine receptor-mediated staining, intracellular ryanodine receptors are found to be localized around the periphery of the cell and the perinucleus region of the cytoplasm (Fig. 4B). Importantly, if cells were treated with an external ligand such as concanavalin A (ConA), the intracellular ryanodine receptors (Fig. 4, D and F) become preferentially associated with surface receptor-patched (Fig. 4C) and -capped (Fig. 4E) structures. Since ConA is known to be involved in T cell-mediated lymphocyte activation and mitogenesis(35) , these results suggest that ryanodine receptor could be critically important for internal Ca release at the onset of receptor capping and lymphocyte activation.


Figure 4: Double immunofluorescence staining of ryanodine receptor- and surface ConA-patched/capped structures. A, staining of ryanodine receptor on the surface of cells (without any ConA treatment or ethanol permeabilization) using rhodamine-labeled mouse monoclonal anti-ryanodine receptor antibody (note that no label was detected). B, staining of intracellular ryanodine receptor of cells (without any ConA treatment but with ethanol permeabilization) using rhodamine-labeled mouse monoclonal anti-ryanodine receptor antibody. C, surface ConA-patched structures induced by fluorescein-labeled ConA. D, intracellular ryanodine receptor staining of cells (with ConA treatment and ethanol permeabilization) using rhodamine-labeled mouse monoclonal anti-ryanodine receptor antibody in the same ConA-patched cells shown in C. E, surface ConA-capped structures induced by fluorescein-labeled ConA. F, intracellular ryanodine receptor staining of cells (with ConA treatment and ethanol permeabilization) using rhodamine-labeled mouse monoclonal anti-ryanodine receptor antibody in the same ConA-capped cells shown in E.



Regulation of Ryanodine Receptor by the Cytoskeletal Protein, Ankyrin

Cytoskeletal proteins are included in the array of cellular elements which underlie and interact with cellular membranes (plasma membrane and/or internal vesicle membranes). It is believed that the formation of a direct connection between the cytoskeleton and certain membrane proteins is one of the earliest events to occur during signal transduction(36) . In fact, a number of cytoskeletal proteins, which are analogs of the major erythrocyte membrane skeleton proteins (e.g. ankyrin, fodrin (a spectrin-like protein), and band 4.1-like protein), are found to be involved in transmitting regulatory signals during cell activation by agonists(37) . Ankyrin is a cytoskeletal protein known to be linked to certain membrane proteins, such as erythrocyte band 3 (37) and lymphocyte GP85(CD44)(38, 39, 40, 41, 42) , and to spectrin/fodrin-actin-associated microfilaments. In addition, ankyrin has been found to bind to the IP receptor in brain tissue (43) and lymphoma cells(13, 44) , and the binding of ankyrin to the IP receptor modulates IP binding and internal Ca release(13, 44) .

In order to determine the binding interaction between lymphoma ryanodine receptor and the cytoskeletal proteins such as ankyrin, we have incubated lymphoma 30 S ryanodine receptor-coated nitrocellulose sheets with I-ankyrin in the presence of various concentrations of unlabeled ankyrin (Fig. 5A). Our data indicate that specific, high affinity binding occurs between ankyrin and this lymphoma 30 S ryanodine receptor (K = 0.075 nM) (Fig. 5A). In addition, we have shown that the binding of I-ankyrin to various 30 S ryanodine receptors isolated from skeletal muscle (Fig. 5B, a), cardiac muscle (Fig. 5B, c), and brain tissue (Fig. 5B, e) is also specific because 100-fold excess soluble unlabeled ankyrin can successfully compete with I-ankyrin binding to these ryanodine receptors (Fig. 5B, b, d, and f). These data suggest that the amino acid sequence of the ankyrin binding domain on various ryanodine receptors must be highly conserved among different cell types and tissues. Recently, we have shown that an 11-amino-acid synthetic peptide derived from sequence comparisons between two ankyrin binding proteins (CD44 and IP receptor) inhibit the binding of ankyrin to mouse lymphoma IP receptor(44) . Preliminary data indicate that this 11-amino acid synthetic peptide (containing the ankyrin binding domain) also blocks ankyrin binding to purified lymphoma 30 S ryanodine receptor with an apparent inhibition constant (K) of 0.25 nM.


Figure 5: Binding of I-ankyrin to various purified 30 S ryanodine receptors. A, purified lymphoma 30 S ryanodine receptor was incubated with I-ankyrin in the presence of various concentrations of unlabeled ankyrin in binding buffer as described under ``Materials and Methods.'' K was measured as 50% inhibition of specific I-ankyrin binding in which the background level of nonspecific binding (i.e. radioactivity detected in the presence of 100 nM unlabeled ankyrin) was subtracted. B, purified 30 S ryanodine receptor isolated from skeletal muscle (a and b), cardiac muscle (c and d), and brain tissue (e and f) was incubated with I-ankyrin in the absence (a, c, and e) and the presence (b, d, and f) of a 100-fold excess amount of unlabeled ankyrin in binding buffer.



Most importantly, the binding of ankyrin (but not fodrin) to the ryanodine receptor in the light density vesicles also significantly inhibits ryanodine binding (Table 3) and blocks the inhibitory effect of ryanodine on internal Ca release (Fig. 6, panel 2). Therefore, it is possible that ankyrin binding competes or overlaps with ryanodine binding sites. This may explain ankyrin's overriding effect on ryanodine-mediated inhibition of internal Ca release in these vesicles (Fig. 6, panel 2). Alternatively, ankyrin binding may cause some conformational change in the regulatory domain(s) of ryanodine receptor resulting in a decrease in ryanodine binding as well as a reduction in the potency of ryanodine inhibition of internal Ca release. The fact that ankyrin also modulates IP binding and IP-mediated Ca release in mouse T-lymphoma cells (13, 44) strongly suggests that ankyrin is one of the important molecules regulating Ca signaling. Currently, we are using in vitro mutagenesis and deletion mutation techniques to further define the ankyrin binding domain(s) on the mouse T-lymphoma ryanodine receptor. In conclusion, our findings support the notion that mouse T-lymphoma cells contain ryanodine receptors which function as a second intracellular Ca release channel along with the IP receptors. Most importantly, we believe that both cADPR and the cytoskeletal protein, ankyrin, play a pivotal role in the regulation of ryanodine-sensitive channel-mediated internal Ca release during lymphocyte activation.




FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM 36353 and CA 66163, a Department of Defense grant, and American Heart Association-Florida Affiliate grants. 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.

§
To whom correspondence and reprint requests should be addressed: Dept. of Cell Biology and Anatomy, University of Miami Medical School, 1600 N. W. 10th Ave., Miami, FL 33101. Tel: 305-243-6985; Fax: 305-545-7166.

The abbreviations used are: RYR, ryanodine receptor; IP, inositol 1,4,5-trisphosphate; cADPR, cADP-ribose; PMSF, phenylmethylsulfonyl fluoride; ConA, concanavalin A; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of Dr. Gerard J. Bourguignon in the preparation of this manuscript.


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