Kinetic Analysis of Inositol Trisphosphate Binding to Pure InositolTrisphosphate Receptors Using Scintillation Proximity Assay

Sandip Patel,* Alison Harris,† Gerry O’Beirne,† Neil D. Cook,† and Colin W. Taylor*,1

*Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, United Kingdom;and †Amersham International, Whitchurch, Cardiff, CF4 7YT, Wales

Received March 22, 1996

Inositol 1,4,5-triphosphate (InsP3) receptors are regulated by many intracellular signals including proteins andsmall messengers. By linking purified cerebellar InsP3 receptors to scintillation proximity assay beads, bindingof radioligands can be measured without separation of bound from free ligand. InsP3 receptors assayed byscintillation proximity assay bound heparin with high affinity and stereoselectively bound InsP3 with similaraffinity to cerebellar membranes. By rapidly freezing scintillation proximity assay reactions and then countingthe frozen samples, both fast and slow components of [3H] InsP3 association and dissociation were identified.Our novel freeze-quench method in combination with conventional stopped-quench equipment and scintilla-tion proximity assay allows the rapid kinetics of the interactions of pure receptors with their ligands to beresolved. © 1996 Academic Press, Inc.

The second messenger inositol 1,4,5-triphosphate (InsP3) links receptor activation at the plasmamembrane to the release of Ca2+ from intracellular stores (1). The InsP3 receptor, a large tetramericCa2+ channel (2), is regulated by the coordinated actions of accessory proteins (e.g., ankyrin,FK-binding protein, calmodulin) (3–5), protein kinase-mediated phosphorylations (6) and diffus-ible messengers (e.g., InsP3, Ca

2+, ATP) (7). Many of these ligands are likely to bind withrelatively low affinity, the kinetics of their binding may be important determinants of the response(7), and the various binding sites are likely to influence each other. Conventional radioligandbinding methods, constrained by the need to separate bound from free ligand, are ill-suited toanalyses of either the rapid kinetics of ligand binding or equilibrium binding of low affinity ligands.In Scintillation Proximity Assays (SPA), receptors can be attached to small beads containing

scintillating fluor. Since the energy of electrons emitted during decay of [3H]ligands is absorbedwithin a fewmm in aqueous media, an SPA bead detects only those [3H]ligands bound to its surface(8). SPA therefore allows receptor-ligand interactions to be detected without the need to separatebound from free ligand. In the present study, we have established an SPA for purified cerebellarInsP3 receptors and used it in a novel way to examine the rapid kinetics of the interactions betweenInsP3 and its receptor.

MATERIALS AND METHODS

Surfact-Amps-X-100 was from Pierce and Warriner Ltd (Chester, U.K.). [3H]Ins(1,4,5)P3 (31 Ci/mmol) and wheat germagglutinin-coated SPA beads were from Amersham International plc.. Heparin (molecular weightz3000) was from Sigma.All other reagents were from the suppliers listed previously (9).InsP3 receptors were purified from rat cerebella by sequential heparin and concanavalin A affinity chromatography as

previously described (9). The receptors were resuspended (z40 mg protein/ml) in binding buffer containing 50 mMTris-HCl (pH 8.3), 1 mM EDTA and 0.1% Surfact-Amps-X-100 and coupled to wheat germ agglutinin-coated SPA beads(40 mg/ml) by incubation at 2°C for 2.5 hours. The receptor-beads were then washed by centrifugation (1 min, 20,000 g)and resuspended (40 mg beads/ml) in binding buffer. Equilibrium-competition binding studies to the receptor-beads (10 mgbeads/ml) were performed in the presence of [3H]InsP3 (5 nM) at 2°C. Bound [3H]InsP3 was determined by countingsamples (200ml) for 60 s in a microfuge tube immersed in iced water using a Packard TriCarb 4530 liquid scintillationcounter. Non-specific binding (typically 10% of total binding) was determined in the presence of 1mM InsP3.

1 To whom correspondence should be addressed. Tel./fax: (+)44 1223 334058.

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For kinetics studies, samples were incubated with either 5 or 10 nM [3H]InsP3 and then rapidly frozen in liquid nitrogenat appropriate times after initiation of association or dissociation reactions. Association reactions were initiated by additionof [3H]InsP3, and dissociation was determined after addition of 1mM unlabelled InsP3 to incubations that had attainedequilibrium (5 min). The microfuge tubes containing the frozen samples were then counted in dry ice-methanol baths for30 s.Equilibrium binding of [3H]InsP3 to cerebellar membranes (10) and to purified receptors (9) was performed using

centrifugation assays as previously described. Equilibrium-competition binding data were fitted to a four-parameter logisticequation using least-squares curve-fitting (Kaleidagraph, Synergy Software, PA):

B 4T − N

1 + H[InsP3#IC50

JnH+ N

where B is the amount of [3H]InsP3 bound; T and N are the total and non-specific binding; IC50

is the concentration of unlabelled InsP3 causing half-maximal displacement of [3H]InsP3, and nH

is the Hill coefficient.

RESULTS AND DISCUSSION

Equilibrium Binding of InsP3

Incubation of purified cerebellar InsP3 receptors with wheat germ agglutinin-coated SPA beadsresulted in coupling of 97 ± 4% (n4 7) of the receptors to the beads, and the coupling was stablefor at least 12 hours at 2°C. InsP3 bound with high affinity to a single class of sites (Kd 4 9.2 ±0.7 nM, nH 4 1.1 ± 0.2, n4 3; Fig. 1A) on the receptor-beads. [3H]InsP3 binding to SPA beadsalone was <5% of that to receptor-beads. These results from SPA are similar to those obtainedusing centrifugation assays of equilibrium binding of InsP3 to cerebellar membranes (Kd 4 8.7 ±1.5 nM, nH 4 0.82 ± 0.15, n4 10) or purified receptors (Kd 4 9.1 ± 2.5 nM, nH 4 1.3 ± 0.3,n 4 5; Fig. 1A inset).L-Ins(1,4,5)P3 (1mM) displaced only 5.2 ± 3.8% (n4 4) of the specific [3H]InsP3 binding from

the receptor-beads, confirming the stereospecificity of InsP3 binding (Fig. 1A). Heparin, a com-petitive antagonist of InsP3 receptors (11), displaced [

3H]InsP3 with similar potency from receptor-beads (IC50 4 90 ± 33 ng/ml, nH 4 1.6 ± 0.2, n4 3) and from cerebellar membranes (IC50 4232 ± 9 ng/ml, nH 4 1.1 ± 0.16, n4 3; Fig. 1B). The ligand-binding properties of InsP3 receptorsare, therefore, similar whether assayed by conventional or SPA equilibrium binding methods.

FIG. 1. Specificity of InsP3 binding to receptor-beads. InsP3 receptor-beads were incubated for 5 min at 2°C with[3H]InsP3 (5 nM) in the presence of varying concentrations of (A) unlabelled D-Ins(1,4,5)P3 (v) of L-Ins(1,4,5)P3 (■) and(B) heparin (v). The inset to A shows [3H]InsP3 (0.5 nM) binding to purified InsP3 receptors in the presence of varyingconcentrations unlabelled D-Ins(1,4,5)P3. Results are the means ± s.e.m. from 3–5 independent experiments.

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Kinetics of InsP3 Binding

Since receptor-ligand interactions can be characterised by SPA without the need to separatebound from free radioligand, SPA allows radioligand association and dissociation to be recordedas they occur. Figure 2A shows the time-course of association of [3H]InsP3 (5 nM) to receptor-beads determined by repeated counting of a single incubation at different times after addition of[3H]InsP3 at 2°C. The results demonstrate that equilibrium binding is attained withinz1 min. Thekinetics of InsP3 association (and dissociation; not shown) are, therefore, too rapid to be charac-terised on-line using SPA. Instead, at appropriate times, samples were rapidly frozen in liquidnitrogen and then counted while still frozen. Freezing did not affect the affinity of the receptor forInsP3 as determined by equilibrium-competition binding (not shown). With this novel method, thekinetics of association and dissociation of [3H]InsP3 (5 nM) to the receptor-beads could be resolved(Fig. 2B).Logarithmic transformation of the kinetics results indicates that the rapid phases of [3H]InsP3

association (Fig. 3A) and dissociation (Fig. 3B) were essentially complete within 4 s (the earliesttime of sampling), and comprised 37 ± 4% (n4 4) and 48 ± 3% (n4 4) of the binding,respectively. From the single exponential describing [3H]InsP3 association after 4 s, the observedrate constant (kobs) was calculated as 0.028 ± 0.003 s

−1 (n4 4). From similar analysis of the slowphase of [3H]InsP3 dissociation, the first order dissociation rate constant (k−1) was 0.02 ± 0.005

−1

(n 4 4). When the radioligand concentration was increased to 10 nM, kobswas 0.036 ± 0.005 s−1

(n 4 3) and k−1 was 0.02 ± 0.001 s−1 (n 4 3).

Time-Dependent Conformational Changes in InsP3 Receptors

Equilibrium binding of InsP3 identified a single class of InsP3-binding site (Fig. 1A), whereasthe kinetics of InsP3 binding were more complex (Fig. 2–3). These differences are likely to reflectconformational changes in the InsP3 receptor following InsP3 binding. The fast phase of InsP3association, which was complete within 4 s (Fig. 3A), is commensurate with the time-course ofInsP3-induced Ca2+ mobilisation (12,13) and is likely to reflect InsP3 binding to an active con-formation of the receptor. The slower phase of association and the binding site that predominatesat equilibrium may then reflect the behaviour of an inactive conformation of the InsP3 receptor. Aprevious study of [3H]InsP3 binding to cerebellar microsomes identified only single kinetic com-

FIG. 2. Kinetics of InsP3 binding to receptor-beads. (A) InsP3 receptor-beads were incubated at 2°C with 5 nM[3H]InsP3 either with (V) or without (v) 1 mM unlabelled InsP3. The samples were repeatedly counted for 10 s atsuccessive intervals in ice/water baths. Results from a single experiment, representative of at least 5 similar experiments,are shown. (B) Association of [3H]InsP3 was initiated by addition of InsP3 receptor-beads (t4 0) to parallel incubationscontaining 5 nM [3H]InsP3. Samples were rapidly frozen at intervals thereafter and counted while still frozen. Dissociationof [3H]InsP3 was measured following addition of unlabelled InsP3 (1 mM) after equilibrium binding had been attained (t4 5 min) and reactions were again terminated by rapid freezing. Results, shown as percentages of specific [3H]InsP3binding at equilibrium, are means ± s.e.m. of 4 independent experiments.

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ponents of association and dissociation (14). In those experiments, where [3H]InsP3 was allowedto bind for onlyz2 s before its removal and measurement of dissociation, the incubation with InsP3may have been too brief to allow the conformational transitions detected using our SPA method.Our results are, however, similar to those previously observed in membranes from liver (15), wherea fast phase of [32P]InsP3 dissociation was complete within 5 s, and a slower phase (k−1 40.03−0.06 s−1) occurred with similar kinetics to those observed by SPA of pure cerebellar receptors(k−1 4 0.02 s−1). In adrenal cortical membranes too, equilibrium binding of [3H]InsP3 identifieda single class of sites, while dissociation kinetics were biphasic (16).

Applications of SPA to Pure Receptors

We conclude that purified InsP3 receptors retain their ability to bind ligands with appropriatespecificity and affinity after attachment to SPA beads. SPA methods can, therefore, be used tocharacterise the interactions between low affinity ligands and pure InsP3 receptors and, using ournovel rapid quenching method, to examine the rapid kinetics of InsP3 receptor-ligand interactions.In a previous study, continuous superfusion of cerebellar membranes immobilised on a filter wasused to examine the rapid kinetics of InsP3 association and dissociation (14). The method is,however, limited by the need to correct for trapped volume, the difficulty of ensuring uniform fluidflow over the membranes, and the very substantial cost of continuous fast superfusion with mediacontaining relatively high concentrations of radioligands. In addition, this method is not readilyapplicable to solubilised receptors. SPA in combination with our freeze-quenching method andconventional stopped-quench technology, provides a more versatile and less expensive means ofexamining the rapid kinetics of the interactions of pure receptors with their ligands.

ACKNOWLEDGMENTS

We thank Dr. J. M. Young for his helpful comments and Ruth Franks for providing rat cerebella. This work wassupported by grants from the Welcome Trust and MRC. C.W.T. is a Lister Institute Research Fellow. S.P. was supportedby a studentship from the BBSRC.

REFERENCES

1. Berridge, M. J. (1993)Nature361,315–325.2. Monkawa, T., Miyawaki, A., Sugiyama, T., Yoneshima, H., Yamamoto-Hino, M., Furuichi, T., Saruta, T., Hasagawa,

M., and Mikoshiba, K. (1995)J. Biol. Chem.270,14700–14704.3. Bourguignon, L. Y. W., and Jin, H. (1995)J. Biol. Chem.270,7257–7260.4. Cameron, A. M., Steiner, J. P., Sabatini, D. M., Kaplin, A. I., Walensky, L. D., and Snyder, S. H. (1995)Proc. Natl.

Acad. Sci. USA92, 1784–1788.5. Yamada, M., Miyawaki, A., Saito, K., Yamamoto-Hino, M., Ryo, Y., Furuichi, T., and Mikoshiba, K. (1995)Biochem.

J. 308,83–88.

FIG. 3. Biphasic kinetics of InsP3 binding. Logarithmic transformation of the association (A) and dissociation (B) resultsshown in Fig. 2. Beq and B are the amounts of [3H]InsP3 bound at equilibrium and at times after initiation of [3H]InsP3association, respectively.

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6. Ferris, C. D., Huganir, R. L., Bredt, D. S., Cameron, A. M., and Snyder, S. H. (1991)Proc. Natl. Acad. Sci. USA88,2232–2235.

7. Taylor, C. W., and Traynor, D. (1995)J. Membr. Biol.145,109–118.8. Bosworth, N., and Towers, P. (1989)Nature341,167–168.9. Richardson, A., and Taylor, C. W. (1993)J. Biol. Chem.268,11528–11533.10. Oldershaw, K. A., Richardson, A., and Taylor, C. W. (1992)J. Biol. Chem.267,16312–16316.11. Taylor, C. W., and Richardson, A. (1991)Pharmac. Ther.51, 97–137.12. Champeil, P., Combettes, L., Berthon, B., Doucet, E., Orlowski, S., and Claret, M. (1989)J. Biol. Chem.264,

17665–17673.13. Meyer, T., Wensel, T., and Stryer, L. (1990)Biochemistry29, 32–37.14. Hannaert-Merah, Z., Coquil, J-F., Combettes, L., Claret, M., Mauger, J-P., and Champeil, P. (1994)J. Biol. Chem.269,

29642–29649.15. Rouxel, F. P., Hilly, M., and Mauger, J-P. (1992)J. Biol. Chem.267,20017–20023.16. Challiss, R. A. J., Chilvers, E. R., Willcocks, A. L., and Nahorski, S. R. (1990)Biochem. J.265,421–427.

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