Rapid Kinetics of myo-Inositol Trisphosphate Binding and ... kinetics of [3H]inositol 1,4,5- ... direct measurements of the rate of InsP, binding to its receptor.
THE JOWALOF B I O ~ I C A CHEMISTRY L. 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 47, Issue of November 25, pp. 29642-29649, 1994 Printed in U.S.A.
Rapid Kineticsof myo-Inositol Trisphosphate Binding and Dissociation in Cerebellar Microsomes* (Received for publication, August 3, 1994, and in revised form, September 21, 1994)
Zalika Hannaert-MerahSQ, Jean-Francois Coquiln, Laurent Combettesn, Michel Claretn, Jean-Pierre Maugefl, and Philippe ChampeilS From the $Unit6 de Recherche Associie 1290 (Centre National de la Recherche Scientifique) et Section de Biophysique des Protiines et des Membranes, Dipartement de Biologie Cellulaire et Moliculaire (Commissariata I’Energie Atomique), Centre &Etudes de Saclay, 91191 Gif-sur-Yvette Cedex, France, llUniti de Recherche UZ74 (Znstitut National de la Sante et de la Recherche Midicale), Universiti Paris-Sud, 91405 Orsay Cedex, France, and SLaboratoire de Biologie de 1’Ecole Centrale de Paris, 92295 ChBtenay-Malabry Cedex, France
Using sheep cerebellum microsomes adsorbedfilon a et al., 1987; Pietri-Rouxel et al., 1992). Second, binding measter,wemeasuredthe kinetics of [3H]inositol 1,4,5- urements have up tonow been restricted t o times longer than trisphosphate (InsP,) binding and dissociation on the a few seconds. As a result, littleis known about the kineticsof subsecond time scale during rapid perfusion of the filter InsP, interaction withits receptor. Studying how rapidly InsP, with [SHIInsP,-containing or InsP,-free media.At 20 “C triggers Ca2+ fluxes has alreadyprovided indirect indicationsof and pH 7.1, in a cytosol-likemedium containing MgCl,, these kinetics (Champeilet al., 1989; Meyer et al., 1990; Ogden the half-time for InsP, dissociation was as short as 125 et al., 1990; Parker and Ivorra, 1990; Finch et al., 1991), but ms. The receptor behaved as a simple target for binding direct measurementsof the rateof InsP, binding to its receptor of its ligand, with the rate constant for InsP, binding are lacking. increasing linearly with InsP, concentration. Various A few previous reports addressed this issue, using manual modulators of InsP, binding (KCl, NaCl, pH, M e , and filtration or ion exchange techniques on the second or minute Ca2+) were found to affect the receptor’s apparent affintime scale (Kingorani et al., 1991; Pietri-Rouxel et al., 1992; ity for InsP, mainly by altering the rate constant for Ribeiro-do-Valle et al., 1994). In the present work, we used a [3H]InsP,dissociation.ATP (but not InsP,) also accelerated [SH]InsP, dissociation. In contrast to these modula-filtration assay for [3H]InsP, binding to cerebellar microsomal membranes, which allowed us both to evaluate InsP, binding tors, luminal Ca” was found to have no effect on the without subsequent washing and to measure the rateof InsP, amount of microsome-bound [SH]InsP,. binding or dissociation on the subsecond time scale under various conditions. At 20 “C under otherwise physiological conditions, the half-time for [3H]InsP, dissociation was a few hunStimulation of various cells by appropriate agonists triggers dred ms. The rate constant for InsP, binding to its receptor phospholipase C-dependent hydrolysis of phosphatidylinositol increased linearly with InsP, concentration, consistent with a bisphosphate in thecell plasma membrane aswell as concomsimple mechanism for InsP, binding. Variousmodulators of the itant release of a water-soluble cytosolic second messenger, receptor’s affinity for InsP, werefound t o mainly actvia moduinositol 1,4,5-trisphosphate (InsP,).’ One of the well known lation of the InsP,dissociation rate. Incidentally, luminal Ca2+, Ca“ effects of released InsP, is to open channels in the cellular another putative modulator of the InsP,receptor (Imine, 19901, storage compartments (reviewed in Berridge (199311, thus iniwas found t o have no influence on InsP, binding under our tiating a rise in the cytosolic free Ca2+.Correspondingly, intraexperimental conditions. cellular membranous receptors to which InsP,bindshave EXPERIMENTALPROCEDURES been isolated, These receptors behave as Ca2+channels when theyarereincorporatedintoartificialmembranes(Ehrlich Sheep cerebella (Institut National de la RechercheAgronomique, andWatras, 1988; Ferris et al., 1989; Maeda et al., 1991; Jouy en Josas) were trimmed, cut in halves, and frozen in liquid nitroMayrleitner et al., 1991; Kamata et al., 1992; Callamaras and gen. Subsequently,halves were ground and homogenized in 45 ml each of a medium containing250 mM sucrose, 5 m M Hepes, 10 mM KC1, 1mM Parker, 1994). p-mercaptoethanol,10 pg/ml leupeptin, 10 PM pepstatin A, 0.2 mM pheThe InsP, binding properties of microsomal fractions ob- nylmethylsulfonyl fluoride, pH 7.4, at 4 “C. The homogenate was centained from different tissues have been extensively studied. trifugedtwice at 1,000 x g (5 min). Supernatants werepooledand This has allowed characterization of the receptors present in centrifuged twice for 10 min (Kontron H 401 Centricon centrifuge, A these fractions (Baukal et al. (1985), Worley et al. (19871, and 8.24 rotor, 10,000 rpm). Supernatants were again centrifuged for 75 min review in Taylor and Richardson (1991)). Nevertheless, a few (Kontron T 2050 centrifuge,TFT 65.38 rotor, 36,000rpm). Pelletswere still poorly docu- finally resuspended in the initial buffer at about 10-20 mg/ml, homogareas of these binding measurements are enized with a Thomas Potter, frozen, and stored in liquid nitrogen. mented. First, most protocols for binding measurements inInsP, binding, measured under control conditions, was reduced by up to clude subsequent washingof free ligand,which might result in 10-20% over 2 h following thawing. This small time-dependent drift a partial loss of bound InsP,if InsP, dissociation is fast (Worley was corrected for. Membranes were thawed and diluted in buffer A(100 m M KCl, 20 mM 5 mM MgCl,, and 25 mM Hepes-KOH pH 7.1) containing 1 m M * The costs of publication of this article were defrayed in part by the NaC1, dithiothreitol and 10 pg/ml leupeptin and kept on ice. Microsome loadpayment of page charges. Thisarticle must therefore be hereby marked “advertisement”in accordancewith 18 U.S.C.Section 1734 solely to ing withCa2+was performed essentially as described before (Combettes et al., 1994). To measure the equilibrium level or the initial rate of indicate this fact. 50-200 pg of protein in 1 The abbreviations used are: InsP,, inositol 1,4,54risphosphate; ko8 binding of [3H]InsP, (DuPont NEN, NET-91), ml was adsorbed onto a Millipore HA filter (0.45 pm). The adsorbed and k,,, dissociation rate constant and bimolecular association rate constant for InsP, binding, respectively; k,,, observed rate constant;Kd, protein was then perfused with an appropriate volume (0.2-3 ml) of binding medium, containing 10 nCi/ml [3HlInsP,and various concenequilibrium dissociation constant.
29642
Rapid Filtration Studiesof PHIInsP, Binding
29643
I I I I 1 trations of total InsP, (at least 0.5 nM; nonradioactive InsP, was from Calbiochem). 100PM [32P]Pi(10 nCilm1) was also included inthe perfu300 2 O " C , pH 8 . EDTA (no KCI) - 3300 sion medium (the[3H]InsP, and [32P]Pi-containing medium was filtered InsP,=0.5 nM through Millex-GS filters to remove undesired aggregates). Radioactivity on the filter was estimated by double counting. The filter itself retained proportional amountsof the two radioisotopes, corresponding to the 30-50 pl of fluid wetting the filter. This wetting volume varied 100 slightly and dependedon the areaover whichthe solution had diffused, but itdid not dependon the presenceof microsomes, as judgedfrom radioactivity. In the presence of microsomes, 32Pradioactivity allowed 0 0 us to precisely determine this wetting volume and the resulting filterI I I I I trapped [3H]InsP3 (about330-550 cpm of 3H; closed symbols inFig. 1). 0 1 2 3 4 This was subtracted from the total [3H]InsP3 on the filter to obtain perfusion p e r i o d , s a microsome-bound [3H11nsP,. Protein was fully retained on the filter, F: I I I I I since protein intrinsic fluorescence was not detectable in the filtrate 5 (see Combettes et al., 19941, and microsome-bound PHlInsP, depended 200 linearly on the amountof microsomes in theassay. Bound [3H]InsP, was expressed as an equivalent volume of the [3H11nsP,-containing solution Dissociation in pl to make it easier to evaluate, by comparison with the wetting volume, if the subtraction procedure contributed a large error. This 100 equivalent volume was directly proportional to the radioactivity expressed in cpm (Fig. 1).When the rate of InsP, dissociation was measured, [3H]InsP, binding was first permitted, as described above (see figure legends) and wasfollowed by perfusion with nonradioactive dis0 sociation medium. In a few cases, binding of [3HlInsP, was also measI I I I I ured after incubation, not perfusion (Fig.3 legend). Experiments were 0 1 2 3 4 performed a t 0 (ice-cold solutions in thecold room), 5, or 20 "C. This protocol with no washing step requires that neither [32PlPi nor p e r f u s i o np e r i o d , s [3HlInsP, adsorb on the filters. This is the case when Millipore HA FIG. 1. Kinetics of [3H]InsP3binding and dissociation at pH 8, filters are used, but it might be useful to emphasize that filters should in the absence of KC1 or divalent cations at 20 "C. A, kinetics of not be soaked together with their spacers in water. This procedure microsomes adsorbed on a nitrocellulose should be avoided because an unknown substance elutesfrom the spac- I3H1InsP, binding to cerebellar filter. The binding medium contained 50 mM Tris-C1 (pH 8,20 "C), 200 ers during soaking and is responsible for [3H11nsP, adsorption on the PM EDTA, and 200 PM EGTA, plus 0.5 nM [3HlInsP3 and 100 PM 132PlPi. filter during subsequent perfusion (data not shown). This adsorption Perfusion periods up to 2 s were controlled with the Biologic rapid can be eliminated by previous soaking of the Millipore HA filters and filtration system. A 4-s perfusion period was obtained by manually spacers in a 1 : l mixture of ethanol with 50 mM Tris-C1, 0.2 mM EDTA, perfusing the adsorbedmicrosomes with 1 ml of binding buffer. [32PlPi and 0.2 mM EGTA a t pH 8, followed by rinsing with water. Undesired radioactivity was used to evaluate the volume of fluid trapped in the adsorption of [3H]InsP, to the filters also occasionally occurred when filter and, therefore, the contribution of this trapped volume (closed glass fiber filters (WhatmanGF/C) were used (data not shown). symbols)tothe L3H1 radioactivity on thefilter. Microsome-bound Rapid filtration was performed with the Biologic rapid filtration unit [3H11nsP, was obtained by subtraction (open symbols). Trapped and (Dupont, 1984; DupontandMoutin, 1987; OrlowskiandChampeil, bound [3HlInsP, are both expressed either incpm (right scale)or in pl, 1991). In this apparatus, a step motor-operated pistonforces the fluid of as an equivalent volume (left scale). The open triangle a t time 0 correthe perfusion syringe through the filter, which is undercontrolled dif- sponds to filters perfused with[3H11nsP,-containing medium for 0.2 or ferential pressure. To avoid favoring preferential pathways inside the 4 s in the absence of microsomes. B , kinetics of [3H]InsP, dissociation filter for the perfusion fluid,we routinely checked that the rateof fluid from cerebellar microsomes. First, binding wasallowed by a 2-s manual delivery by the syringe corresponded to the maximal rate a t which fluid perfusion of adsorbed microsomes with 0.5 ml of binding medium. Time 0 correspondstosuchsamples,countedwithout any washing step. could flow through the filter.' by perfusing the filter for Measurements were generally performed in triplicate, and the mean[3HlInsP, dissociation was then triggered various periods with the same medium now in theabsence of [3HlInsP3 -c S.D. was plotted. The dissociation and binding kinetics were fitted to and [3zP]Pi.Open and closed symbols as for panel A. Fit to singleexpoa simple exponential behavior, usinga SigmaPlot routine (JandelScinentials was as follows: kobs(in 5-l) was 0.65 f 0.15 (open triangles in of entific). To fit the equilibrium binding measurements in the presence and 0.25 * 0.03 (open circles in panel B ) . increasing concentrationsof unlabeled InsP,, an equation implying spe- panel A ) cific binding to the receptor (witha Hill coefficient n ) plus nonspecific additional rinse. Microsome-bound [3H]InsP, was determined binding, and corrected for thevariable specific activity of total by subtracting the amount of [3H]InsP3in the filter wet volume [3HlInsP,, was used.
y = (B,,J(l+ (K,/C)") + aC) * (C,/C)
(Eq. 1)
B,,, is the maximalspecific binding, Kd the dissociation constant,C the free concentration of InsP,, including the [3H11nsP3tracer, n the Hill coefficient, 01 the factor describing nonspecific binding, and C,,the free concentration of the [3H11nsP, tracer. RESULTS
Measurement of Equilibrium fH]InsP, Binding withoutSubsequent Washing-To measure [3HlInsP, binding without subsequent washing, we adapted a protocol previouslyusedto study binding of ligands to high affinity sites on sarcoplasmic reticulum ATPase (Dupont, 1980; Yamaguchi and Watanabe, 1989; Orlowski and Champeil, 1991). Briefly, membranes suspended in an Imp,-free medium were adsorbed on a nitrocellulose filter; [3H]InsP, was allowed to bind to its receptor duringcontinuous perfusion of thefilterwithan [3H]InsP,containingmedium;andthefilterwas counted with no F. Guillain and P. Champeil, unpublished observations.
from the total [3H]InsP, on the filter. [3H]InsP, per se did not adsorb on the filters (see "ExperimentalProcedures"; see also open triangles at time 0 in Figs. 1-3 and diamonds inFigs. 3 0 and 4, C and D ) . In the presence of microsomes, virtually all microsome-bound [3HlInsP, was displaced by excess nonradioactive InsP,, so that 90-99% of microsome-bound [3H]InsP, at low [InsP,] represented specific binding to the InsP, receptor (see Figs. 3 0 and 4, C and D ) . Under favorable conditions, [3HlInsP, binding could be measured both with our perfusion protocol and by a standard method, ie. by incubating [3H]InsP, with microsomes in a test tube before filtration, with identical results (see Fig. 3 legend). When a short washing step, after binding, was included in the protocol (e.g. washing with 1or 2 ml of ice-cold EDTA-containing sucrosemedium at pH 7.4, which lasted 2-3 s), 2 0 4 0 % of the previously bound [3H]InsP3 was lost, irrespective of the procedure used for binding (see also Figs. 2 and 3). This justifies our present attempt to measure [3HlInsP, binding without any subsequentwashing. Kinetics of PHlInsP, Binding andDissociation-The kinetics of [3HlInsP, binding to cerebellar microsomes were first meas-
Rapid Filtration Studies of PHIInsP, Binding I
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FIG.3. Kinetics of [SH]InsP3 binding anddissociation at various total concentrationsof InsP,, in the absenceof KC1 or divalent cations atpH 8 and 5 "C. The binding and dissociation medium contained 50 mM Tris-C1,200 PM EDTA, and 200 PM EGTA, and various concentrations of nonradioactive InsP, (pH8 . 0 , 5 "C). Triangles inpanel A correspond to t3H11nsP, binding in the presenceof 60 nM t3HlInsP,. Circles in panel A correspond to [3H11nsP, dissociation in a medium containing the same concentration of nonradioactive InsP,; in this case, prelirninaly binding took place for 30 s in a test tube, so that equilibrium was reached before perfusion started. Panel B shows binding measurements performed at a lower t3HlInsP3 concentration, 0.5 nM. For the latter experiment, the amount of protein layered ontothe filter was 4-fold smaller than for the experiment illustrated in panelA; the as an final amount of bound [3HJInsP, was higher when expressed equivalent volume, in pl, but was of course lower when expressed in absolute numbers(pmoVmgof protein). I n both panelsA and B , the 30-s data points were obtained after incubating microsomes with InsP, in a test tube. Fit to single exponentials wasfollows: as k,,, (in 5-l) was 1.35 * 0.30 (triangles) or 0.14 2 0.02 (circles) in panel A and 0.19 2 0.02 (squares) in panel B. C , observed rate constantfor I3H1InsP3binding, as a function of the total [3HJInsP, concentration (triangles). Circles refer to the dissociation rates measured in the presence of various concentrations of nonradioactive InsP,. D,Kd for equilibrium binding.Various 10 nCi/rnl concentrations of nonradioactiveInsP,wereaddedto [3HlInsP, tracer, and binding to themicrosomes was measured after a 30-s incubation in a test tube. Squares and triangles correspondtwo to of proteins different experiments, performed with different amounts and normalized. The line corresponds to nH= 1 and Kd = 8 n ~ plus , a very small contribution of nonspecific sites. The diamond in panel D corresponds to a filtration measurement performed in the absence of microsomes.
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FIG.4. Equilibrium 13H]InsP3 binding under various ionic conditions at20 "C. This series of experiments was performed at 20 "C. Adsorbed microsomes were perfused for 2 s with 0.5 ml of a binding medium containing200 p~ EDTA and 200 I.~MEGTA in all cases.A, the medium also contained0.5 nM total InsP, and either50 mM Tris-C1 (pH 8.0) (squares) or100 rnM KCl, 20 mM NaCI, and 25 mM Hepes-KOH a t various pH, in the absenceof Mg2' (circles)or in the presence of 5 mM Mg2' (triangles). B , the medium contained 0.5 n~ InsP,, 100 mM KCl, 20 mM NaCl, 25 mM Hepes-KOH (pH 7.4), plus various concentrations of M e . The resulting free M e is indicated on the abscissa. C , the medium contained 100 mM KCl, 20 mM NaCl, 25 mM Hepes-KOH (pH 7.41, and various total concentrations of InsP,. D,The medium contained 100 mM KC], 20 mM NaCl, 25 m~ Hepes-KOH, 5 mM MgZ' (pH 7.1) and, again, various total concentrations of InsP,. For the experiment illustrated in panel D, microsomes were twice as concentrated as for the other panels. In panels C and D,diamonds correspond to filtration measurements performed in the absence of microsomes. The lines in panels C and D are fits to theoretical binding with nH= 1 and Kd= 38 and 170 nM, respectively, with nonspecific binding corresponding to less than 5 1.11.
(square in panel A ) . The receptor's affinity for InsP, was studied under two conditions, namely at pH 7.4 in the absence of M e (panel C ) and at pH 7.1 in the presence of 5 mM Mg2f (panel D ) (KC1 and NaCl being present in both cases; for the experiments performed at pH 7.1 in thepresence of MgZ' (panel Dl, the protein concentration during the measurements was increased to make the resultsmore reliable). Dissociation constants were 45 * 15 nM (average of two experiments; panel C illustrates one of them) and170 50 IIM (pane2 D), respectively, with Hill coefficients close to 1 (1.03 t 0.03). In all cases the maximal binding capacity deduced from our fits was of the order of 25 5 pmol [3HlInsP, bound per mg of protein, as previously found (Hilly et al., 1993). Results were therefore number of sites and if this affinity change is mainly derived consistent with the idea that changes in Kd (not changes in the from a change in the dissociation rate constant. This was fur- maximal binding capacity) were mainly responsible for changes ther investigated. in the amountof bound [3H]InsP3.Similar inhibitory effects of Fig. 4 first documents the modulating effect of pH, M e , KCI, pH and M e have been previously described (Worley et al., and NaCl on the amountof I3HIInsP, bound after a 2-s manual 1987; Joseph et al., 1989; Volpe et al., 1990; White et al., 1991; perfusion with 0.5 nM [3H]InsP,. As experiments wereperVan Delden et al., 1993). Note that the large amountof bound formed at 20 "C, 2-s perfusion was virtually sufficient to reach [3HlInsP, previously found at 0 "C (Fig. 223) could well be also equilibrium (e.g. see Fig. 2 A ) . The amount of [3H]JnsP, bound associated with a Kd lower than those measured at 20 "C (comunder these conditions depended greatly on the ionic medium. pare Fig. 30 with Fig. 4 , C and D). Thiseffect of temperature Acidic pH was inhibitory as well as MgZ' (Fig. 4,panel A ) .At pH has also been described previously (Pietri-Rouxel et al., 1992). ~ and 20 mM NaC1, the 7.4 and in the presence of 100 r n KC1 We then investigated the effect of these different media on concentration of Mg2' for 50% reduction of bound [3H]InsP, was the kinetics of InsP, dissociation. Thus, Fig. 5A shows that the in the millimolar range (panel B ) . At pH 8 and in theabsence presence of KC1 and NaCl accelerated [3H]InsP, dissociation by of M e , removal of KC1 and NaCl enhanced [3H]InsP, binding a factor of 2-3 compared with a Tris-C1 medium (circles versus
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Rapid Filtration Studies of PHlInsP, Binding
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perfusion p e r i o d , s FIG.5. Kinetics of [3H11nsP, dissociation after a KC1 or a pH jump. These experiments were performedat 20 "C. Allmedia contained 200 1.1~EDTA, 200 1.1~EGTA, and 10 nM total InsP,. [3HlInsP3binding took placeduring manual perfusion for 2 s prior to dissociation.A, effect ofKC1 and NaC1. Both the binding and dissociation media contained either 50 mM Tris-C1at pH 8.0 (squares, "Zkis"medium) or 100 nm KC1, 20 mM NaC1, and 25 IIU~Hepes-KOH at pH 8.0 (circles, "KC? medium). Panels B and C , KC1 and NaCl jump. The pH 8 media used for binding and dissociation were now different, respectively: Zkis-Zkis (squares) or Zkis-.KCl (invertedtriangles) in panel B ; KC1-KC1 (circles) or KCl+Dis (triangles) in panel C . D , pH jump. The binding and dissociation media both contained 100 mM KC1,20 mM NaC1, and 25 rn Hepes-KOH. Binding took place at pH 8.0 in both cases. Dissociation took place either at pH 8 (circles) or pH 6.8 (diamonds). Fit to single exponentials was as follows: k,, (in s-l) was 0.54 t 0.12 (squares),1.5 f 0.3 (circles),1.6 f 0.3 (inuerted triangles),0.25 f 0.05 (triangles),or 4.5 z 1(diamonds).
squares), inagreement with the lower levelof [3HlInsP, bound at equilibrium (time 0) in thepresence of these ions. However, since extending these measurements to other conditions with low amounts of [3HlInsP3bound at equilibrium would become increasingly difficult, we considered the possibility of first allowing r3H1InsP, to bind to microsomes under favorable conditions and then monitoring the rate of [3HlInsP, dissociation under different conditions. Such "jumps" to the final ionic medium have been previously used to measure the rate of dissociation of ligands from sarcoplasmic reticulum ATPase under various conditions (e.g. de Meiset al. (1980) and Orlowski and Champeil(1991)). We first checked under favorable conditions the reliability of this procedure. Thus, Fig. 5B shows that after [3H]InsP3binding in the absence ofKC1, addition ofKC1 and NaCl to the dissociation medium did accelerate [3HlInsP3dissociation (triangles versus squares). Conversely,panel C in the same figure shows that after I3H1InsP3binding in thepresence of KC1 and NaCl, omission of these ions from the dissociation medium slowed down [3H]InsP3dissociation (triangles versus circles). On this basis, we then tested the effect of pH on the dissociation rate by first allowing t3H]InsP3to bind to the adsorbed microsomes at pH 8 and then measuring the rate of [3H11nsP, dissociation in a pH 6.8 medium versus a pH 8 medium. The more acidic pHinduced faster dissociation, consistent with the lower levelof [3HlInsP3bound at equilibrium (Fig. 5 0 , compare with Fig. 4A).Using the same protocol, we found that Mg2' also accelerated t3H]InsP3dissociation (Fig. 6A),consistent with the
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FIG.6. Effect of Ca2+, nonradioactive InsP,, and ATP on [3HlInsP,binding and dissociation. These experiments were performed a t 20 "C. Allmedia contained 100 mM KC1,20 mM NaC1,25 mM Hepes-KOH, 200 EDTA, 200 EGTA, and 10 1 1 ~total InsP [3HlInsP3binding took place during manual perfusion for 2 s. A, Ma' jump. The binding and dissociation media were buffered at pH 7.4. Dissociation took placeeither in the absence of Mg2' (circles)or in the presence of a 5 mM MgZ' (triangles). Panels B-D, effect of Ca2+,ATP, and excess nonradioactive InsP, on equilibrium binding of [3HlInsP,,(panel B ) and on [3HlInsP,dissociation rate (panels Cand D ) . The binding and dissociation media werebuffered at pH 8. B , equilibrium [3HlInsP, binding under control conditions or with 500 total Ca" Le. 100 p~ free Ca''), 1.5 II~MNafiTP, or 1.5 p~ nonradioactiveInsP,. Panels Cand D , Ca", ATP, and InsP, jumps. After [3HlInsP3binding under control conditions, t3H1InsP, dissociation took place either under control conditions (circles) or in the presence of the same concentrations of Ca2+ (diamonds in panel C ) ,NafiTP, or nonradioactive InsP, (triangles and squares in panel D,respectively)as those used in the experiment illustrated in panel B . Fit to single exponentials was as follows: kabs(in s-') was 1.3 t 0.25 (circles)or 5.6 1.2 (triangles) in panel A and 0.95 f 0.2 (circles),5 t 2 (diamonds), 2.4 f 0.7 (triangles),or 1.2 f 0.2 (squares)in panels C and D.
observed reduction of equilibrium [3H]InsP3binding. Incidentally, these marked effects of an ionic jump during dissociation imply that the receptor relaxed to its new state more rapidly than [3HlInsP3dissociated from the initial state, prerequisite a for such jump experiments. These measurements were extended to other previously documented situations that affect [3H]InsP3 binding, namely the presence of excess Ca" or millimolar concentrations ofATP, both known to reduce [3HlInsP3binding t o cerebellar microsomes (e.g. Worley etal. (1987),Joseph et al. (1989),and Nunn and Taylor (1990); see Fig. 6 B ) . A Ca2+jump (Fig. 6C) clearly accelerated [3HlInsP, dissociation. An ATP jump (Fig. 6D, triangles versus circles) also did so, although t o a proportionally smaller extent than the extent of binding inhibition (see "Discussion"). In contrast, the presence of excess nonradioactive InsP, did not modify the rateof dissociation of previously bound 2 0.2 [3HlInsP,significantly (Fig. 6D, squares uersus circles; 1.2 versus 0.95 2 0.2 s-'). Finally, we aimed at using our [3H]InsP3binding protocol to study how previous loading with Ca2+of the cerebellar microsomes altered the affinity of their receptors for InsP,. It was previously suggested that increasing the Ca2+content of Ca2+ stores in A7r5 cells increased the sensitivity of InsP,-induced Ca2+release to InsP,, at least until the CaZ+content reached
29647
Rapid Filtration Studiesof PHlInsP, Binding
TABLEI1 TABLEI Summary of the equilibrium dissociation constantsand dissociation Effect of luminal Ca2' on PHlInsP, binding to cerebellar microsomes rate constants measured under various conditions, with the 50 pl of microsomes were incubatedfor 4 min at 20 "C with 250 pl of corresponding calculated bimolecular association rate constants buffer A containing 60 p~ EGTA and 10 PM added Ca2+ (plus tracer al. et (1994)) in the 45Ca2+ for Caa+ uptake measurements; see Combettes Dissociation Calculated association absence or presenceof 1.5 mM ATP and a regenerating system (loading Experimental dissociation rate constant rate constant conditions constant medium). Microsomes were then diluted in buffer A containing 1 mM Kd koR EGTA; for some samples, this medium also contained 0.5 p~ InsP,, known to completely empty the InsP,-sensitive store (quenching medinM s" 107 M" s-1 um). 10-15 s later, microsomes were adsorbed onto Millipore HA filters, 5 "C rinsed with2 ml of a medium containing250 mM sucrose, 25 mM Hepes- Tris, pH 8, EDTA 8 f 3" 0.16 f 0.02" 2-cl Tris, 200 pv EDTA, 200 pv EGTA, and 100 V M Pi (pH 7.1,20 "C), and (2.4 5 0.8 measured)" finally perfused again (this lasted about 3 s ) with another 2 ml of the 20 "C same sucrose medium containing 0.5 n~ InsP,. For [3H11nsP3binding Tris, pH 8, EDTA 11 -c 3' 0.40 f 0.18' 3.6 f 2.6 measurements, the latter medium also contained [3H11nsP3and [32P3Pi KCI, pH 8, EDTA 29 -c 9' 1.5 c 0.4d 5.2 f3 tracers. The indicated result is the mean of four measurements,f S.D. KC1, pH 7.4, EDTA 45 c 15" 1.65 f 0.85f 3.7 * 3 Additional control experiments with 45Ca2+-loadedmicrosomes showed KC1, pH 7.1, Mg 170 2 509 5.7 f 2' 3.3 2 that (i)perfusion of the loaded stores with 0.5 nM InsP, for 3 s did not induce any measurable 45Caa+release (the finalload was 1.66 k 0.03 in a Fig. 3. the presence of InsP,, compared with 1.67 k 0.02 in its complete abData not shown. sence); (ii) perfusionfor 3s with the high concentrationof 0.5 pv InsP, Figs. 1 and 5. in thesucrose medium inducedonly minimal releaseof 45Ca2+ (the final Fig. 5. load was 1.57 f 0.03 uersus 1.67 i 0.02);(iii) in contrast, perfusion for e Fig. 4C and data not shown. 3 s with 0.5 p~ InsP, in a medium identical t o the KCI-containing f Figs. 2A and 6 A . g Fig. 4. quenching medium induced close to 100% release of 45Ca2+(the final load was 1.12 k 0.01, compared with 1.13 -c 0.01); (iv) in the absence of Data not shown (freeMgZ' was 3.7 mM). of the KC1-containing InsP,, perfusion withthe sucrose medium instead medium had no significant effect (final loads were1.67 -c 0.02 or 1.61 c 0.04, respectively). thus, this protocol also allowed us to perform experiments at KO,
*
'
20 "C and in the presence of Mg2' or Ca2+,two conditions known tostimulatethese activities. Usingthis protocol, [3HlInsP, binding and 45Ca2+ release (Combettes et al., 1994) could also be nmol "Ca2'lmg protein Pl measured under exactly the same conditions (e.g. see Table I). 124 -c 9 No ATP/EGTA 0.08 f 0.01 ATPEGTA 1.66 0.03 119 -c 4 The fast response of InsP,-dependent channels to thepresence ATP/EGTA + InsP, 1.13 2 0.02 115 = 10 of their activating ligands makes such measurements of the initial bindingof InsP, particularly relevant. Kinetics of PHJInsP, Binding and Dissociation-The kinetics about 30% of thesteadystate value (Paryset al., 1993). [3H]InsP, binding was measured here withtotally empty com- of [3H]InsP, binding and dissociation were monitored on the partments or compartments that had been loaded with Ca2+by subsecond time scale (Figs. 1-3, 5 and 6). Experiments perprevious incubation with ATP or, again, with microsomes that formed under favorable conditions (Tris-C1 medium, alkaline pH, and low temperature; see Fig. 3) showed that theclassical had initially been loaded with Ca2+but whose InsP,-dependent compartments had been subsequently emptied by addition of equations describing interaction of a ligand L with its target content of were valid for the InsP, receptor (Equations 2-4; see "Results"), InsP, (which is a control for empty pools). The 45Caz+ thestores was measuredin parallel. Table I shows that i.e. the observed rate constantfor ligand binding is equal to the [3HlInsP, binding was virtually identical in all three cases dissociation rate for very low ligand concentrations and increases ina linear way for higher ligand concentrations (see for tested, i.e. binding was independent of luminal Ca2+in the instance Fig. lD in Champeil et al. (1989)). To the contrary, InsP,-sensitive compartments. Equation 2 implies that there is no additional information in DISCUSSION the measurementof the rateof [3H]InsP, binding at low [InsPJ, Measurements of PHIInsP, Binding withoutSubsequent compared with the measurement of its dissociation rate; new Washing--To measure InsP, binding tocerebellar membranes, information can only be obtained from specially dedicated exwe used a protocol in which, after dilution of the microsomes periments performed at InsP, concentrations of the sameorder and adsorption on a filter, [3HlInsP, was allowed to bind to its of magnitudeasthe equilibrium dissociation constant or receptor duringperfusion of the filter witha medium contain- higher, as shown in Fig. 3. Note that one of the early studiesof ing [3HlInsP, and the desired additives. Then the filter was the InsP, receptor reported that the rateof InsP, dissociation counted with no rinsing. Theabsence of a rinsing step ensured was faster than its rate of binding (Baukal etaZ., 1985), behavthat all InsP, binding sites were titrated, notonly those whose ior that is not compatible with a simple mechanism for binding dissociation half-times were long enough to withstand rinsing. (Equation 2). Nevertheless, we will assume here that Equation Subtraction of the background signal, correspondingto thevol- 1, demonstrated under selected conditions (Fig. 3, C and D ) , ume of fluid trapped in the filter, from the totalradioactivity on also holds under different conditions. the filter was particularly reliable because of the high density A k,, of 2 * lo7 M-'s-' was measured at 5 "C (Fig. 3C). Table of InsP, receptors in cerebellar microsomes; nevertheless, a I1 summarizes values measured for the dissociation rate consimilar subtraction alreadyproved to be feasible also in a less stant and the equilibrium constant under different ionic confavorable case, using liver membranes (Pietri-Roue1 et al., ditions at 20 "C as well as the corresponding bimolecular rate 1992). This protocol, coupled with electronic control of the per- constants for InsP, binding, which can be deduced therefrom, fusion period by the Biologic rapid filtration system, allowed us according to Equation3. The measuredor calculated k,, values here to study the kinetics of [3H]InsP, binding and dissociation. all fall in the 2-5 * lo7 "l5-l range, corresponding to secondAs microsomes were continuously perfused with the [3H]InsP,- order rate constants slower than diffusion-controlled and typcontaining medium, any InsP, molecule degraded because of ical of many enzyme ligands (e.g. Hiromi, 1979). They are of the microsomal phosphatases or generated because of microsomal same order of magnitude as previous indirect estimates based phospholipases was immediately washed off, and theadsorbed on the kinetics of InsP,-dependent Ca2+fluxes (Champeil et al., microsomes remained in contact with fresh perfusion medium; 1989; Meyer et al., 1990). Turning to the implications of our Loadindquenching
45Caz+ content
L3HIInsP, bound (equivalent volume)
29648
Rapid Filtration Studiesof PHlInsP, Binding
results under "physiological" conditions,but being restricted t o 20 "C, the half-time for [3H]InsP, dissociation in thepresence of KC1 and NaCl at pH 7.4 was about 400 ms (Figs. 2A and 5 A ) . It was reduced t o about 125 ms in thepresence of M e , both at pH 7.4 and 7.1 (Fig. 6A and Table 11). The latter conditions correspond t o previous 45Ca2+ flux measurements in our laboratory (Combettes et al., 19941, conditions under which a Kd value of 170 nM was measured (Fig. 40).Using Equations 2 and 3, this implies that thehalf-time for InsP, binding will beabout 60 ms at 170 nM and 15-20 ms at 1 p~ InsP,. Thus, binding of InsP, per se will generally not greatly delay cellular response. Atlow temperatures, very low concentrations of [,H]InsP, equilibrated slowly with our cerebellar microsomes, namely over seconds.Yet, the binding and dissociation rates we measured were faster than some of those previously reported. From a practical point of view, this implies that previous studies of InsP, binding in whichmicrosomeswere first rinsed before filter counting may have ignored part of the bound InsP,. Nevertheless, it is also possible that different receptors have different characteristics from the point of view of binding and dissociation rates (e.g. Guillemette et al. (19871, Challis et al. (19901,Kamata et al. (1992),Pietri-Rouxelet al. (19921,Mohr et al. (19931, and Ribeiro-do-Valleet al. (199411, either because of different conformations or because of different structures. In fact, one potentially important aspect of the present work is to provide a general method to discriminate, on the basis of their kinetics, between the various types of receptors that have been identified (Furuichi et al., 1989; Siidhof et al., 1991; Ross, et al., 1992; Blonde1 et al., 1993). It could alsobe useful to analyze the effect of the interaction of InsP, receptor with other intracellular proteins like ankyrin, a cytoskeletal protein (Bourguignon et al., 1993; Joseph and Samanta,1993).However, such studies will only be possible with tissues with a reasonably high density of receptors. On the basis of similar dissociation measurements performed on a slower time scale with membranous fractions from hepatocytes, Pietri-Rouxel et al. (1992) observed biphasic dissociation kinetics at low temperatures, which were attributed to the existence of two different forms of receptor. In thiswork, the data points for the kinetics of either [3HJInsP,binding or dissociation were fitted reasonably well with single exponentials, but insome cases the fit was not perfect (e.g. Figs. 5 and 6 ) . Unresolved biphasicity might also be the reason why binding and dissociation could not always be fitted to single exponentials with exactly the same half-time (Figs. 1 and 2). This could be because of slow isomerization steps. Alternatively, although the type l InsP, receptor was found t o be the predominant species in mouse cerebellum (Furuichi et al., 19931, we cannot exclude the possibility that other isoforms are expressed in sheep cerebellum and partly account forthis additional complexity. Exploring these hypotheses will require a great deal more data than collected here to make possible detailed analysis in terms of biphasic kinetics. Modulation of PHIInsP, Binding underVarious ConditionsOur results confirm previous suggestions that changes in affinity for InsP, of the receptor are at least partly responsible for the effects of various modulators of [3H]InsP, binding like KC1 and NaC1, MgZ+,pH, or Ca". In addition, our time-resolved results show that modulation of the rate of InsP, dissociation plays a prominent role in these affinity changes. Our finding of a Ca2+-inducedchange in the rateof [3H]InsP, dissociation (Fig. 6 C ) excludes the possibility that theinhibitory effect of 100 p~ Ca2+could be entirely caused by activation of phospholipases (Mignery et al., 1992). ATP also affectedthe [3HlInsP, dissociation rate (Fig. 6 D ) , although t o a smaller extent than expected on the basis of the concomitant reduction in equilibrium bind-
ing (Fig. 61, confirming that nucleotides suppress bound [3HlInsP, not only by competition for the same site but also by binding to separate sites, which modify the InsP, receptor (Bezprozvanny and Ehrlich, 1993). The observation that 1.5 JIM nonradioactive InsP, did not accelerate [3HlInsP, dissociation significantly (Fig. 6 D ) is consistent with little cooperative interaction, from the point ofviewof ligand binding, between InsP, molecules boundt o adjacent monomers in the tetrameric receptor, at least under our assay conditions. Finally, the absence of effect of luminal Ca2+on [3H]InsP, binding is worth special comment.Previously published results obtained with different systems were indicative of a small effect of luminal Ca2+(Parys et al., 1993; Oldershaw and Taylor, 1993). In the present measurements, we followed the suggestion made by Oldershaw and Taylor to measure [3H]InsP3binding not only under conditions oflow [3H]InsP, but also in a KC1-free medium, i.e. under conditions where InsP, binds but does not trigger Ca" release efficiently (we confirmedthis; see Table I legend). This minimized not only the average Ca2+loss by the Ca2+pools but also the local changes in luminal Ca2+ close to those few receptors with bound [3HlInsP,. Nevertheless, no effect of the Ca2+load was observed (Table I). Before abandoning the idea that luminal Ca2+controls the InsP, receptor, however,the question arises of whether those receptors in our cerebellar membranes that bind [3H11nsP3are thesame as those t o which InsP, binds to trigger Ca2+release. Although the InsP, dependence previously foundby us for the kinetics of 45Ca2+ release from microsomes (Fig. 1OB in Combettes et al. (1994)) is compatible with the Kd of 170 2 50 nM found in this work under the same conditions (Fig. 401, we cannot answer this question in a completely unambiguous way. In fact, many different receptors certainly exist, not all of which have the same affinity, localization, and therefore functional role (e.g. Furuichi et al. (19931,Joseph and Samanta(19931, Khodakhah and Ogden (19931, and Kuno et al. (1994)). Alternatively, the putative effect of luminal Ca", if present, might result from an increase in the probability of the channel being open rather than from a change in the receptor's affinity for InsP,. Acknowledgments-We thank A. W. Boydfor help in editing the manuscript andDrs. C. W. Taylor and S. Swillens for critical comments. REFERENCES Baukal, A. J., Guillemette, G., Rubin, R., Spat, A., and Catt, K. J. (1985)Biochem. Biophys. Res. Commun. 133, 532-538 Bemdge, M. J. (1993) Nature 361, 315-325 Bezprozvanny, I., and Ehrlich, B. E. (1993) Neuron 10, 1175-1184 Blondel, O., Takeda, J., Janssen, H., Seino, S., and Bell, G. I. (1993)J. Biol. Chem. 268, 11356-11363 Bourguignon, L. Y. W., Jin, H.,Iida, N., Brandt, N. R.,and Zhang, S. H. (1993) J. BLOC. Chem. 268,7290-7297 Callamaras, N., and Parker, I. (1994) Cell Calcium 15, 66-78 Challis, R. A. J., Chilvers, E. R., Willcocks, A. L., and Nahorski, S. R. (1990) Biochem. J. 265,421427 Champeil, P., Combettes, L., Berthon, B., Doucet, E., Orlowski, S., and Claret, M. (1989) J . Bid. Chem. 264. 17665-17673 Combettes, L., Hannaert-Merah,Z., Coquil, J. F., Rousseau, C., Claret, M., Swillens, S., and Champeil, P. (1994) J . Biol. Chem. 269, 17561-17571 de Meis, L., Martins, 0. B., and Alves, E. W. (1980) Biochemistry 19, 42524261 Dupont, Y. (1980) Eur. J . Biochem. 109,231-238 Dupont, Y. (1984)Anal. Biochem. 142, 505-510 Dupont, Y., and Moutin, M. J. (1987) Methods Enzymol. 148, 675-683 Ehrlich, B. E., and Watras, J. (1988) Nature 336,583-586 Ferris, C . D., Huganir, R. L., and Snyder, S. (1989)Nature 3 4 2 , 8 7 4 9 Finch, E. A,, Turner, T. J., and Goldin, S. M. (1991) Science 252, 443-446 Furuichi, T.,Yoshikawa, S.,Miyawaki,A., Wada, K., Maeda, N., and Mikoshiba, K. (1989) Nature 342, 32-38 Furuichi, T., Simon-Chazottes, D., Fujino, I., Yamada, N., Hasegawa, M., Mivawaki,A., Yoshikawa, S., GuBnet, J.-L.,and Mikoshiba,K. (1993)Receptors and Channels 1, 11-24 Guillemette, G., Balla, T., Baukal, A. J., Spat, A., and Catt, K. J. (1987) J . Biol. Chem. 262,1010-1015 Hilly, M., Pietri-Rouxel,F., Coquil, J. F., Guy, M., and Mauger,J.-P. (1993) J. Biol. Chem. 268,16488-16494 Hingorani, S. R., and Agnew, W. S. (1991)Anal. Biochem. 194, 204-213 Hiromi, K. (1979) Kinetics of Fast EnzymeReaction:Theory and Practice, pp. 255-273, John Wiley & Sons, Inc., New York ~
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