IntroductionThe cytosolic Ca2+ concentration ([Ca2+]c) controls, throughvarious Ca2+ signalling pathways, essential and diverse cellularprocesses, such as cell division and apoptosis, in addition toproviding a major trigger for smooth muscle contraction(Horowitz et al., 1996; Whitaker and Larman, 2001; Berridgeet al., 2003). [Ca2+]c is crucially affected by the activity of theintracellular store (the sarcoplasmic reticulum, SR), whichregulates Ca2+ release (Bootman et al., 2001; Berridge et al.,2003; McCarron et al., 2004b). In many tissues, one of the twomajor routes of Ca2+ release from the SR is the inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] receptor (IP3R), the other one isthe ryanodine receptor (RyR). Central to an understanding ofCa2+ signalling therefore, is an appreciation of the control ofIP3R-mediated Ca2+ release (Hanson et al., 2004; Patterson etal., 2004; Taylor et al., 2004).

IP3R activity is regulated by accessory proteins such ascalmodulin (Taylor and Laude, 2002; Kasri et al., 2002), certainneuronal Ca2+-binding proteins (Yang et al., 2002; Kasri et al.,2004) and the anti-apoptotic protein Bcl-2 (Chen et al., 2004).

The 12 kDa FK506-binding protein, FKBP12 also interactswith IP3R to regulate IP3-mediated Ca2+ release (Cameron etal., 1995b; Dargan et al., 2002), athough this has been disputed(Bultynck et al., 2001a; Bultynck et al., 2001b; Carmody et al.,2001). FKBP12 reportedly increases, decreases or has no effecton IP3-mediated Ca2+ release, even when bound to the receptor.With regard to FKBP12’s binding to the receptor, neithersolubilised IP3R nor proteolytic fragments containing IP3Rcombined with glutathione-S-transferase (GST)-FKBP12Sepharose columns, though RyR did so (Bultynck et al., 2001a;Carmody et al., 2001; Zheng et al., 2004). IP3R also did not co-immunoprecipitate or co-purify with FKBP12 in cerebellarmicrosomes (Bultynck et al., 2001a; Thrower et al., 2000). Yet,in other co-immunoprecipitation, co-purification and direct-binding-assay studies, FKBP12 was tightly bound to IP3R in ratcerebellum (Cameron et al., 1995a; Cameron et al., 1995b).Further studies using the yeast two-hybrid system, showed thebinding of FKBP12 to the IP3R and located the site of bindingto the leucyl-proline dipeptide residues 1400-1401 (Cameron etal., 1997).

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Ca2+ release from the sarcoplasmic reticulum (SR) by theIP3 receptors (IP3Rs) crucially regulates diverse cellsignalling processes from reproduction to apoptosis.Release from the IP3R may be modulated by endogenousproteins associated with the receptor, such as the 12 kDaFK506-binding protein (FKBP12), either directly orindirectly by inhibition of the phosphatase calcineurin.Here, we report that, in addition to calcineurin, FKPBsmodulate release through the mammalian target ofrapamycin (mTOR), a kinase that potentiates Ca2+ releasefrom the IP3R in smooth muscle. The presence of FKBP12was confirmed in colonic myocytes and co-immunoprecipitated with the IP3R. In aortic smoothmuscle, however, although present, FKBP12 did not co-immunoprecipitate with IP3R. In voltage-clamped singlecolonic myocytes rapamycin, which together with FKBP12inhibits mTOR (but not calcineurin), decreased the rise incytosolic Ca2+ concentration ([Ca2+]c) evoked by IP3Ractivation (by photolysis of caged IP3), without decreasing

the SR luminal Ca2+ concentration ([Ca2+]l) as did themTOR inhibitors RAD001 and LY294002. However,FK506, which with FKBP12 inhibits calcineurin (but notmTOR), potentiated the IP3-evoked [Ca2+]c increase. Thispotentiation was due to the inhibition of calcineurin; it wasmimicked by the phosphatase inhibitors cypermethrin andokadaic acid. The latter two inhibitors also prevented theFK506-evoked increase as did a calcineurin inhibitorypeptide (CiP). In aortic smooth muscle, where FKBP12 wasnot associated with IP3R, the IP3-mediated Ca2+ release wasunaffected by FK506 or rapamycin. Together, these resultssuggest that FKBP12 has little direct effect on IP3-mediatedCa2+ release, even though it is associated with IP3R incolonic myocytes. However, FKBP12 might indirectlymodulate Ca2+ release through two effector proteins: (1)mTOR, which potentiates and (2) calcineurin, whichinhibits Ca2+ release from IP3R in smooth muscle.

Key words: Ca2+ signalling, Smooth muscle, FKBP12, IP3 receptors

Summary

In smooth muscle, FK506-binding protein modulatesIP3 receptor-evoked Ca2+ release by mTOR andcalcineurinDebbi MacMillan, Susan Currie, Karen N. Bradley, Thomas C. Muir and John G. McCarron*Institute of Biomedical and Life Sciences, Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow,G12 8QQ, UK*Author for correspondence (e-mail: J.McCarron@bio.gla.ac.uk)

Accepted 23 August 2005Journal of Cell Science 118, 5443-5451 Published by The Company of Biologists 2005doi:10.1242/jcs.02657

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Results from functional studies of IP3R modulation byFKBP12 are also controversial. The association betweenFKBP12 and IP3R is disrupted by immunosuppressant drugs,such as FK506 and rapamycin, that bind to FKBP12 to formdrug-immunophilin protein complexes, which then displace theaccessory proteins from the channel (Cameron et al., 1995b;Cameron et al., 1997; Dargan et al., 2002). Whereas FK506abolished ATP-induced Ca2+ oscillations in tracheal epithelialcells, thereby suggesting a modulating role for FKBP12 in IP3release (Kanoh et al., 1999), no functional effect of FK506 orFKBP12 on IP3-induced Ca2+ release has emerged in A7r5,SH-SY5Y, C2C12 or COS-7 cells (Bultynck et al., 2001a;Boehning and Joseph, 2000; Bultynck et al., 2000). Evenwhere functional effects of FKBPs in IP3-evoked Ca2+ releasehave been claimed, controversy persists. In some studies,removal of FKBP12 from IP3R increased (Cameron et al.,1995b; Cameron et al., 1997), whereas in others the additionof FKBP12 to IP3R increased the activity of the channel(Dargan et al., 2002). In the former studies, disruption ofFKBP12 binding to IP3R increased the activity of the channel(Cameron et al., 1995b). In the latter study, recombinantFKBP12, when added to the purified cerebellar IP3R1 isoformincorporated into planar bilayers, substantially increased theactivity of the channel and induced ‘coordinated gating’ ofneighbouring receptors (Dargan et al., 2002), an effect reversedby FK506.

One explanation for the apparent controversy might beFKBP12’s ability to regulate IP3R indirectly (Cameron et al.,1995a). Displacement of FKBP12 from IP3R by FK506 resultsin the formation of an FK506-FKBP12 complex that binds toand inhibits the Ca2+/calmodulin-dependent phosphatasecalcineurin (Liu et al., 1991). Indeed, calcineurin inhibitionmight mediate FK506’s immunosuppressant actions (Liu etal., 1992). FKBP12 might localise calcineurin to the IP3R toregulate the phosphorylation status of the channel (Cameronet al., 1995a; Kawamura and Su, 1995). Indeed, in cerebellum,the physical association of calcineurin with the IP3R-FKBP12complex is displaced by FK506 (Cameron et al., 1995a;Cameron et al., 1997). This association increases IP3Rphosphorylation and enhances Ca2+ release (Cameron et al.,1995a). Again, in adrenal glomerulosa cells, both Ca2+

signalling and protein kinase C (PKC)-mediatedphosphorylation of the IP3R were modified by FK506 (Poirieret al., 2001), whereas in COS-7 cells, calcineurin reduced IP3-induced Ca2+ release, an effect reversed by FK506(Bandyopadhyay et al., 2000). However, as with FKBP12, themechanisms by which calcineurin regulates Ca2+ release fromIP3R are disputed. Indeed, calcineurin might regulate Ca2+

release independently of FKBP12 (Bultynck et al., 2003) or,in other cases, not at all (Kanoh et al., 1999). In the latterstudy, in airway epithelial cells, FK506 attenutated ATP-induced Ca2+ oscillations, whereas calcineurin inhibitors didnot.

FKBPs are also displaced from the IP3R by the bacterially-derived antibiotic rapamycin from Streptomyceshygroscopicus (Marks, 2003). Rapamycin was originallyused as an antifungal agent but has since been discardedbecause of its undesirable immunosuppressive side effects.These side effects were subsequently explored and developedand the drug was approved for clinical use as animmunosuppressant (e.g. Marks, 2003; Barshes et al., 2004).

The intracellular receptor for rapamycin is FKBP12 but thecomplex so formed does not inhibit calcineurin (unlike theFK506–FKBP12 complex). The molecular target for therapamycin-FKBP12 complex is the protein kinase ‘target ofrapamycin’ (TOR), its mammalian homologue is calledmTOR (Heitman et al., 1991). mTOR is a phosphatidylinositol-related kinase that is inhibited by the rapamycin-FKBP12 complex. mTOR integrates signals from nutrients(amino acids and energy) and growth factors (in highereukaryocytes) to regulate and coordinate cell growth and cell-cycle progression (reviewed by Panwalkar et al., 2004).Although no direct experimental link between mTOR andIP3-mediated Ca2+ release has been established so far,rapamycin itself reportedly decreased Ca2+ release fromcerebellar microsomes (Dargan et al., 2002) even though theproposed mechanism did not involve mTOR.

In view of the potential importance of FKBP12 inregulating IP3-mediated Ca2+ release, with accompanyingconsequences for Ca2+ signalling and the persistentcontroversy regarding the interaction between IP3R andaccessory proteins the present study was undertaken. Wepropose here, mechanisms by which FKBP12 regulates IP3-evoked Ca2+ release in smooth muscle. Freshly isolated singlecolonic smooth muscle cells were selected; IP3-evoked Ca2+

release does not activate RyRs in this cell type (Flynn et al.,2001; McCarron et al., 2004a), simplifying the analysis ofresults. Cells were voltage-clamped in the whole-cell-configuration to avoid [Ca2+]c changes that might occurthrough Ca2+ influx as a result of changes of the membranepotential, evoked by rapamycin or FK506. The use of flash-photolysis of caged IP3 minimised the activation of secondmessenger systems to give a clearer understanding of thecontrol of Ca2+ release from the receptors. The study foundthat mTOR inhibitors – including rapamycin – that operatethrough FKBP12, inhibited IP3-mediated Ca2+ release.However, calcineurin inhibitors operating through FKBP12,including FK506, increased Ca2+ release; FK506 wasineffective after calcineurin had been blocked. In aorticsmooth muscle, in which FKBP12 did not associate with thereceptor, neither rapamycin nor FK506 altered IP3-mediatedCa2+ release. We propose that, when associated with thereceptor, FKBP12 itself has little direct effect on IP3R butpotentiates Ca2+ release by inhibiting calcineurin or reducesCa2+ release by blocking mTOR.

Materials and MethodsMaterialsCaged Ins(1,4,5)P3-trisodium salt was purchased from MolecularProbes (Leiden, the Netherlands). Fluo-3 penta-ammonium salt waspurchased from TEF Labs (Austin, Texas, USA). Rapamycin,cypermethrin and okadaic acid were each purchased fromCalbiochem-Novabiochem (Beeston, Nottingham, UK), anti-IP3R(type 1) and anti-FKBP12 antibodies from Affinity BioReagents(Golden, Colorado, USA). The anti-calcineurin B antibody (anti-calcineurin/PP2B A beta) was purchased from Upstate (Dundee, UK),RAD001 was a gift from Novartis Pharma AG (Basel, Switzerland)and FK506 a gift from Fujisawa GmbH (Munich, Germany). All otherreagents were purchased from Sigma (Poole, UK). Caffeine (10 mM)dissolved in extracellular bathing solution, was applied withhydrostatic pressure (PicoPump PV 820, World Precision Instruments,Stevenage, UK). The concentration of caged, non-photolysed IP3

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refers to that in the pipette. FK506 and RAD001 each were dissolvedin 100% ethanol [the final bath concentration of the solvent by itself(0.05%) was ineffective]. Rapamycin, cypermethrin, okadaic acid andLY294002 hydrochloride were each dissolved in dimethylsulphoxide[final bath concentration of the solvent by itself (0.01%) wasineffective]. Each drug (with the exception of caffeine) was perfusedinto the solution bathing the cells (~5 ml/minute). The calcineurininhibitory peptide (CiP) based on the autoinhibitory fragment(ITSFEEAKGLDRINERMPPRRDAMP) was obtained from Sigma(Poole, UK).

MethodsCell dissociation

Male guinea pigs were killed by cervical dislocation with immediateexsanguination in accordance with the Animal (Scientific Procedures)Act 1986, UK. Single smooth-muscle cells were enzymaticallyisolated from the guinea-pig colon or aorta, stored at 4°C and used onthe same day (McCarron and Muir, 1999). All experiments wereconducted at room temperature (20-22°C). Cells were voltage-clamped using conventional tight-seal whole-cell recording. Thecomposition of the extracellular solution was: Na-glutamate (80 mM),NaCl (40 mM), tetraethylammonium chloride (TEA) (20 mM), MgCl2(1.1 mM), CaCl2 (3 mM), HEPES (10 mM), and glucose (30 mM)(pH 7.4 adjusted with 1 M NaOH). The pipette solution contained:(Cs)2SO4 (85 mM), CsCl (20 mM), MgCl2 (1 mM), HEPES (30 mM),pyruvic acid (2.5 mM), malic acid (2.5 mM), KH2PO4 (1 mM),MgATP (3 mM), creatine phosphate (5 mM), guanosine triphosphate(0.5 mM), fluo-3 penta-ammonium salt (0.1 mM) and cagedIns(1,4,5)P3-trisodium salt (caged IP3) (0.025 mM) (pH 7.2 adjustedwith 1 M CsOH). Whole cell currents were amplified by an Axopatch1D amplifier (Axon instruments, Union City, CA, USA), low passfiltered at 500 Hz (eight-pole bessel filter; Frequency Devices,Haverhill, MA), and digitally sampled at 1.5 kHz using a Digidatainterface, pCLAMP software (version 6.0.1, Axon Instruments) andstored on a personal computer for analysis.

[Ca2+]c was measured as fluorescence from the membrane-impermeable dye Fluo-3 introduced into the cell through the patchpipette (McCarron and Muir, 1999). To photolyse caged IP3 (25 �M),the output of a xenon flashlamp (Rapp Optoelektronik, Hamburg,Germany) was passed through a UG-5 filter to select UV light andmerged into the excitation light path of the microfluorimeter using thesecond arm of the quartz bifurcated fibre-optic bundle (McCarron andMuir, 1999) and applied to the caged compound. Fluorescence signalswere expressed as ratio (F/F0) of fluorescence counts (F) relative tobaseline (control) values (taken as 1) before stimulation (F0).

Immunoprecipitation and western blottingAll procedures were performed at 4°C. Freshly isolated and hand-homogenised smooth muscle from guinea pig colon or aorta wassolubilised (Cameron et al., 1995a), but using a lower concentrationof Triton X-100 (0.2% Triton X-100 for 60 minutes), which, whencombined with low speed centrifugation (1500 g for 10 minutes),minimised mechanical and chemical inhibition of FKBP interactionswith receptors (Carmody et al., 2001; Dargan et al., 2002; George etal., 2003). IP3R protein was immunoprecipated from 500 �g (totalprotein) samples of this preparation by overnight incubation withrabbit anti-IP3R antibody (Affinity BioReagents, Golden, USA)followed by incubation with protein G-sepharose for a further 30minutes. The sepharose beads were then washed and IP3Rs eluted byheating at 70°C in 4� Laemmli sample buffer.

Sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) wasperformed as described by Currie and Smith (Currie and Smith, 1999),except that 3-8% Tris-acetate gels were used for IP3R and calcineurinA detection, and 12% Bis-Tris gels for FKBP12 and calcineurin Bdetection. Recombinant FKBP12 (for FKBP12 protein) and non-

immunopreciptated solubilised supernatant (for IP3R, calcineurin Aand calcineurin B proteins) served as positive controls. Proteins weredetected with specific rabbit primary antibodies against IP3R,FBKP12, calcineurin B (each from Affinity BioReagents, Golden,USA), mTOR (Cell Signalling Technology Inc., Beverly, USA), andwith mouse monoclonal anti-calcineurin A (Sigma, Poole, Dorset,UK) followed by incubation with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Sigma, Poole, UK). Blots weredeveloped using the enhanced chemiluminescence (ECL) detectionsystem (Amersham Bioscences, Amersham, Bucks, UK). Qentix wasused as a western-blot enhancer for FKBP immunoblots (PierceBiotechnology, Rockford, USA).

Statistical analysisResults are expressed as means ± s.e.m. Student’s t-test was appliedto test and control conditions, a value of P<0.05 was consideredsignificant.

ResultsTo investigate the role of FKBP12 in regulating IP3R activity,the interaction between FKBP12 and IP3R was studied byimmunoprecipitation of IP3R1 (the main isoform in this tissue)from solubilised guinea-pig colon circular smooth-musclehomogenates. IP3R immunoprecipitates were subjected toimmunoblotting and the presence of both IP3R and FKBP12confirmed using specific antibodies (Fig. 1A,B). The bindingof FKBP12 to IP3R was disrupted by either FK506 orrapamycin (Fig. 1C,D). Two of the cellular targets of FKBP12,the serine/threonine protein kinase mTOR (Fig. 1E) and thephosphatase calcineurin (Fig. 1G) were also present in thetissue as revealed by western blotting. Indeed calcineurin – butnot mTOR (data not shown) – also co-immunoprecipitatedwith IP3R (Fig. 1G,I). The association of calcineurin with IP3Rwas not reduced by pre-incubation of the tissue with FK506(20 �M; 2-4 hours, n=3; data not shown) prior to thesolubilization or by incubation of the homogenate with FK506(20 �M; 2-4 hours, n=3; data not shown), suggesting thatcalcineurin-binding to this receptor in this tissue did not requireFKBP12.

To determine whether or not mTOR regulates Ca2+ releasethrough IP3R, the effects of rapamycin, which disrupts theFKBP12-IP3R complex and inhibits mTOR (but notcalcineurin), were examined on IP3-induced Ca2+ release involtage-clamped single smooth-muscle cells. Photolysed cagedIP3 (25 �M) activated IP3R and increased [Ca2+]c. Rapamycin(10 �M) significantly (P<0.05) decreased the IP3-evoked Ca2+

transient (�F/F0) by 42±7% from 1.53±0.41 to 0.79±0.18 (n=7,Fig. 2A). The decrease is unlikely to be explained simply by areduction in the store’s Ca2+ content by rapamycin’s inhibitionof the SR Ca2+ pump (Bultynck et al., 2000), because the Ca2+

transient in response to RyR activation with caffeine wassignificantly (P<0.05) increased by rapamycin – rather thandecreased – by 56±10% (�F/F0 from 0.51±0.12 to 0.77±0.15;n=10), (Fig. 2B). However, inhibition of mTOR might explainthe rapamycin-induced reduction in the IP3-evoked Ca2+

transient. To explore this possibility, the rapamycin analogueand mTOR inhibitor RAD001 (Panwalker et al., 2004; Huangand Houghton, 2003; Majewski et al., 2003) was studied. Thiscompound also decreased IP3-mediated Ca2+ release (Fig. 3A)as did the mTOR and phosphatidylinositol 3 kinase inhibitor

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LY294002 (20 �M) (Brunn et al., 1996) (Fig. 3B). Thus,RAD001 (10 �M) and LY294002 each significantly (P<0.05)decreased IP3-mediated Ca2+ release. RAD001 by 38±6%(�F/F0 from 1.37±0.33 to 0.88±0.23, n=8) (Fig. 3A) andLY294002 by 40±8% (�F/F0 from 2.11±0.43 to 1.37±0.44,n=8) (Fig. 3B). These results suggest that mTOR potentiatesCa2+ release from the IP3R in this tissue and that this effect isreduced by inhibition of mTOR.

In contrast to the results with rapamycin, FK506 (10 �M),which inhibits both the FKBP12-IP3R interaction andcalcineurin but not mTOR (Cameron et al., 1995b; Cameron etal., 1997; Dargan et al., 2002), significantly (P<0.05) increasedIP3-mediated Ca2+ release in voltage-clamped single smooth-muscle cells by 30±10% (�F/F0 from 2.05±0.19 to 2.67±0.31,n=11, Fig. 4). This difference might have occurred becauseFK506, unlike rapamycin, inhibits the Ca2+-activatedphosphatase calcineurin, whereas FK506 and rapamycin eachdisrupt FKBP12 interaction with IP3R1. If so, then the

potentiation of Ca2+ release by FK506 might be mediated bythe inhibition of calcineurin. To examine this, the effects ofcalcineurin inhibition on IP3-evoked Ca2+ release were studied.Photolysed caged IP3 reproducibly increased [Ca2+]c in thesecells. The calcineurin inhibitor cypermethrin (10 �M) and theprotein phosphatase inhibitor okadaic acid (5 �M eachsignificantly (P<0.001 and P<0.05, respectively) increased thisrise in Ca2+ (�F/F0) by 85±23% and 33±12%, from 0.45±0.1to 0.74±0.11 and from 0.85±0.22 to 1.03±0.23, respectively(n=12 and 8, respectively (Fig. 5A and Fig. 5B, respectively),suggesting that calcineurin regulates the phosphorylation stateof the IP3R (Cameron et al., 1995a). Significantly,cypermethrin, okadaic acid and the calcineurin inhibitorypeptide (CiP) each prevented the FK506-induced increase inIP3R-mediated Ca2+ release (Fig. 6A-C). Thus, in the presenceof 100 �M CiP (where CiP was administered into the cellthrough the pipette solution because it is impermeant), FK506(10 �M) did not significantly alter the IP3-evoked Ca2+

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Fig. 1. Co-immunoprecipitation of IP3R and FKBP12 and the presence of mTOR in colonic smooth muscle. IP3R1 (the major isoform present;data not shown) was immunoprecipitated from solubilised colonic smooth muscle. (A,B) Immunoblots were probed with rabbit anti-IP3R1 andrabbit anti-FKBP12 antibodies for the presence of (A) IP3R1 and (B) FKBP12, respectively. Lane 1 in each panel shows theimmunoprecipitated protein from colon. Lane 2 shows the relevant positive control (10 �g solubilised supernatant for IP3R or 50 ngrecombinant FKBP12). Arrows on the right indicate the position of molecular-mass markers run in parallel to indicate protein migration on thegel. The detection of a band at 12 kDa (B) indicates that FKBP12 is present and associated with IP3R1 in these myocytes. These data arerepresentative of four experiments. (C) FKBP12-IP3R1 association in colon is disrupted by FK506 and rapamycin. IP3R1 wasimmunoprecipitated from solubilised colon. Immunoprecipitates were probed for the presence of IP3R1 (C) and FKBP12 (D). Lanes 1-4 in eachpanel are the immunoprecipitated protein from colon, lane 5 is antibody alone (negative control), lane 6 is solubilised colon protein plus IgGsepharose without antibody (negative control) and lane 7 the relevant positive control [solubilised supernatant (C) and recombinant FKBP12(D)]. The detection of a band in untreated control preparations at 12 kDa (D, lanes 3 and 4) indicates that FKBP12 is present and associatedwith IP3R1 (n=2). This FKBP12- IP3R1 association was disrupted by the addition of FK506 (D, lane 1, 20 �M) and rapamycin (D, lane 2, 20�M), each of which reduced the FKBP12 signal. Arrows indicate the position of molecular-mass markers run in parallel to indicate proteinmigration on the gel. (E) mTOR is present in colonic myocytes. Immunoblots were probed with the anti-mTOR antibody (lane 1). Lane 2shows the molecular-mass marker to show protein migration on the gel. (F, G) Co-immunoprecipitation of IP3R1 and calcineurin B fromsolubilised colonic smooth muscle. Immunoprecipitations were performed as above using rabbit anti-IP3R1 antibody, and immunoblots probedfor the presence of IP3R1 (F) and calcineurin B (G). Lane 1 in each panel is the immunoprecipitated protein from colon, lane 2 is solubilisedpreparation plus protein G without antibody, lane 3 antibody alone, lane 4 the positive control (10 �g solubilised supernatant in each case).Arrows indicate the position of molecular-mass markers run in parallel. The detection of a band at 19 kDa indicates that calcineurin B is presentand associated with IP3R1. These data are representative of six experiments. (H, I) Co-immunoprecipitation of IP3R1 and calcineurin A fromsolubilised colonic smooth muscle. Immunoprecipitations were performed as above using rabbit anti-IP3R1 antibody and immunoblots wereprobed for the presence of IP3R1 (H) and calcineurin A (I). Lane 1 in each panel is the immunoprecipitated protein from colon, lane 2 thesolubilised preparation plus protein G without antibody, lane 3 the antibody alone and lane 4 the positive control (10 �g solubilised supernatantin each case). Arrows on the right indicate the position of molecular-mass markers run in parallel. The detection of a band at 55 kDa indicatesthat calcineurin A is present and associated with IP3R1. This data is representative of two experiments.

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transient (�F/F0 from 1.43±0.42 to 1.54±0.46 in the additionalpresence of FK506, n=11, P>0.05; Fig. 6A). Aftercypermethrin, the IP3-evoked Ca2+ increase was also unalteredby FK506 (Fig. 6B; �F/F0 was 1.1±0.17 in cypermethrin and0.99±0.17 in the additional presence of FK506, n=6, P>0.05).After incubation with okadaic acid, the Ca2+ increase was alsounaltered by FK506 (Fig. 6C; �F/F0, 1.94±0.33 in okadaic acidand 1.89±0.38 in the additional presence of FK506, n=7,P>0.05). IP3-mediated Ca2+ release was not maximallyactivated in the presence of the phosphatase inhibitors. Thus,the thiol-reactive agent thimerosal, which potentiates IP3-

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Fig. 2. Effects of rapamycin on IP3- and caffeine-evoked Ca2+ increases in voltage-clamped single colonic myocytes. (A) Photolysed caged IP3(F) increased [Ca2+]c as indicated by F/F0. Rapamycin (10 �M, n=7) significantly reduced (P<0.05) the IP3-evoked [Ca2+]c transients.(B) Rapamycin (10 �M, n=10, P<0.01) significantly increased the Ca2+ rise evoked by caffeine (CAF, 10 mM) following activation of RyR.

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Fig. 3. Effects of the mTOR inhibitors RAD001 and LY294002 on IP3-evoked Ca2+ increases in voltage-clamped single colonic myocytes. ThemTOR inhibitors each inhibited IP3-evoked Ca2+ increases. Photolysed caged IP3 (F) increased [Ca2+]c as indicated by F/F0. The IP3-evoked[Ca2+]c transient was significantly decreased by each inhibitor. (A) RAD001, 10 �M, n=8, P<0.05. (B) LY294002, 20 �M, n=8, P<0.05).

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)Fig. 4. Effects of FK506 on IP3-evoked Ca2+ increases in voltage-clamped single colonic myocytes. Photolysed caged IP3 (F)significantly increased [Ca2+]c as indicated by F/F0. FK506 (10 �M,n=11, P<0.05) potentiated the IP3-evoked [Ca2+]c transients.

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mediated Ca2+ release (e.g. Bootman et al., 1992), increasedIP3-mediated Ca2+ release by a further 14±2% (n=4, P<0.05)after the phosphatase inhibitor cypermethrin. In theseexperiments IP3 evoked a �F/F0 increase of 0.97±0.49 incontrol cells, 1.77±0.63, in cypermethrin (10 �M) alone, and1.99±0.69 in cypermethrin (10 �M) and thimerosal (100 �M)(n=4). Together, these results suggest that FK506 potentiatesIP3-mediated Ca2+ release by inhibition of calcineurin.

In aortic smooth muscle the interaction between FKBP12and IP3R was also studied by immunoprecipitation fromsolubilised guinea-pig aorta homogenates. Here aortic smoothmuscle, FKBP12 was expressed at similar levels to thoseoccurring in colon (Fig. 7A), however it did not co-immunoprecipitate with IP3R (Fig. 7B,C). Also, rapamycin (10�M) or FK506 (10 �M) did not significantly (P>0.05) alterIP3-induced Ca2+ release in voltage-clamped single aortic

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Fig. 5. Effects of the calcineurin inhibitors cypermethrin and okadaic acid on IP3-evoked Ca2+ increases in voltage-clamped single colonicmyocytes. Photolysed caged IP3 (F) increased [Ca2+]c as indicated by F/F0. The IP3-evoked [Ca2+]c transient was significantly increased by eachinhibitor. (A) Cypermethrin 10 �M, n=12, P<0.001; (B) okadaic acid 5 �M, n=8, P<0.05.

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increases following calcineurin inhibition involtage-clamped single colonic myocytes.Following pre-treatment with the calcineurininhibitors (A) CiP (100 �M), (B) okadaic acid (5�M) or (C) cypermethrin (10 �M), FK506 (10�M) did not increase the IP3-evoked [Ca2+]ctransient produced by photolysed caged IP3 (F) asit had done in the absence of inhibitors (cf.Fig. 4).

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smooth-muscle cells (Fig. 7D,E). Thus, �F/F0 was 0.9±0.28 incontrol and 0.86±0.26 in FK506 (10 �M; n=6) and, in separateexperiments, �F/F0 was 1.26±0.55 in control and 1.18±0.52 inrapamycin (10 �M; n=3).

DiscussionIn defining the mechanism by which FKBP12 regulates IP3-evoked Ca2+ release in intact single colonic myocytes, we havefound that FKBP12 coexists in a macromolecular complex withIP3R. However, it apparently has little direct effect on IP3-evoked Ca2+ release but might indirectly either increase ordecrease this release by inhibiting effector proteins. FKBP12might increase IP3R-mediated Ca2+ release indirectly byinhibiting calcineurin or decrease release by inhibiting mTOR.In support, FK506, which disrupts the binding of FKBP12 toIP3R and together with FKBP12 inhibits calcineurin,potentiated IP3-mediated Ca2+ release. Each of the phosphataseinhibitors, cypermethrin and okadaic acid, also potentiated IP3-mediated Ca2+ release. Significantly, after cypermethrin,okadaic acid or CiP, FK506 no longer increased IP3-mediatedCa2+ release. These results suggest that calcineurin is requiredfor FK506-mediated potentiation of IP3-evoked Ca2+ release

and imply that FKBP12 itself might not significantly influencethe regulation of direct Ca2+ release through IP3Rs.

Rapamycin disrupts the binding of FKBP12 to the receptorand together with FKBP12 inhibits mTOR. Like the mTORinhibitors RAD001 and LY294002 (the latter also inhibitsphosphoinositide 3-kinase) rapamycin also reduces IP3-mediated Ca2+ release. These results are unlikely to be due toa reduced luminal SR [Ca2+]l by virtue of SR Ca2+ pumpinhibition (Bultynck et al., 2000; Bilmen et al., 2002), becausethe Ca2+ increase evoked by RyR activation (by caffeine) wasincreased by rapamycin, indicating the adequacy of the Ca2+

content of the SR. In our experiments, IP3R was activateddirectly by photolysis of caged IP3, so obviating the signaltransduction pathway that mediates IP3 synthesis. Theinhibition by rapamycin cannot be explained by an indirectblock of IP3-mediated Ca2+ release caused by an alteredsarcolemma membrane potential (Weidelt and Isenberg, 2000)because all cells were voltage-clamped. A reversal of thepotentiating effect of FKBP12 on IP3R activity by rapamycinas reported by Dargan et al. (Dargan et al., 2002), is alsounlikely to account for the present findings because theremoval of FKBP12 by FK506 had no effect on IP3-mediatedCa2+ release after calcineurin inhibition. Taken together, these

Fig. 7. IP3R and FKBP12 do not co-immunoprecipitate in aortic smooth muscle and neither rapamycin nor FK506 alter IP3-evoked Ca2+

increases in voltage-clamped single aortic myocytes. (A) Similar amounts of FKBP12 protein are expressed in aortic and colonic smoothmuscle. Colon and aorta were each hand-homogenised as described in Materials and Methods, and solubilised supernatants from eachhomogenate was assayed for FKBP12 protein expression. Proteins (10 �g total) from each tissue were separated using SDS-PAGE andimmunoblots were probed for the presence of FKBP12 (lanes 1 and 4). Migration and signal intensity were compared with those obtained fromrecombinant FKBP12 (50 ng) run alongside as positive controls (lanes 2 and 3). The blot is representative of seven and four identicalexperiments, in colonic and aortic smooth muscles, respectively. (B,C) IP3R1 was immunoprecipitated from solubilised aortic smooth muscle.Immunoblots were probed with rabbit anti-IP3R1 and rabbit anti-FKBP12 antibodies for the presence of (B) IP3R1 and (C) FKBP12,respectively. The first lane in each panel shows the immunoprecipitated protein from aorta, lane 2 the solubilzed preparation plus protein Gwithout antibody, lane 3 the antibody alone and lane 4 the relevant positive control (10 �g solubilised supernatant for IP3R or 50 ngrecombinant FKBP12). Arrows on the right indicate the position of molecular mass markers run in parallel to indicate protein migration on thegel. The absence of a band at the 12 kDa level (C) indicates that FKBP12 is not associated with IP3R1 in these myocytes. These data arerepresentative of three experiments. (D,E) Photolysed caged IP3 (F) increased [Ca2+]c as indicated by F/F0. Neither (D) rapamycin (10 �M,n=3) nor (E) FK506 (10 �M; n=6) significantly altered the IP3-evoked [Ca2+]c transients (P>0.05).

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results suggest an important role for mTOR in regulating IP3-mediated Ca2+ release but the mechanisms involved remainunclear.

Our findings show that FKBP12 colocalised with the IP3Rin colonic myocytes, although whether or not this is a directinteraction is unclear. Some reports by other investigatorsconfirm, whereas others dispute, a physical interaction betweenFKBP12 and IP3R. For example, although the type 1 IP3Rcontains a consensus sequence for FKBP12 binding, it failedto immobilise FKBP12 (Bultynck et al., 2001a), suggesting nophysical interaction between them. However, our study andthose of others have shown a physical interaction between IP3Rand FKBP12 (Cameron et al., 1995a; Cameron et al., 1995b).Methodological differences could explain these apparentlycontradictory results. Detergents, high-speed centrifugationand high temperatures have each already been shown to disruptthe interaction between RyR and FKBP (Dargan et al., 2002;George et al., 2003), and this might also apply to the interactionbetween IP3R and FKBPs. The position of a cell in its life cycle(Bultynck et al., 2001a) or its signalling status (Carmody et al.,2001), e.g. the extent of phosphorylation of the IP3R – as withFKBP12.6 and RyR (Marx et al., 2000) – might also determinewhether or not FKBP12 is bound to IP3R. Moreover, speciesor tissue differences might also provide an explanation for thevariation in results. Indeed, in this study, in experimentalconditions in which FKBP12 co-immunoprecipitated withIP3R in colon cells, no association between the receptor andFKBP12 was seen in aortic smooth muscle. Furthermore,neither rapamycin nor FK506 altered IP3-mediated Ca2+

release in intact cells from this tissue, suggesting that absenceof association was not a result of a disrupted interactionbetween FKBP12 and IP3R caused by co-immunoprecipitationmethods.

In this study, calcineurin also co-immunoprecipitated withIP3R in colonic myocytes. However, the colocalization ofcalcineurin does not appear to require FKBP12 because FK506did not reduce the binding of calcineurin to IP3R. In colonicmyocytes, unlike in brain membranes (Cameron et al., 1995b),FKBP12, calcineurin and IP3R might not exist in a trimericcomplex.

The IP3R is phosphorylated by multiple serine/threonineprotein kinases, including cAMP-dependent protein kinase andPKC, to increase the activity of the channel (reviewed byPatterson et al., 2004). The serine/threonine protein kinasemTOR is a member of the phosphoinositol kinase-relatedkinase family (reviewed by Panwalkar et al., 2004) andinvolved in the regulation of cell growth by initiating genetranslation in response to nutrients such as ATP, amino acids(mainly leucine), growth factors, insulin and mitogens. mTORmight also be involved in a diversity of additional cellularfunctions, including actin organization, secretion, membraneactivity and PKC signalling (reviewed by Panwalkar et al.,2004). The present results imply that, among its multiple anddiverse effects, mTOR also regulate IP3Rs. This activity mightbe supported by the localization of the kinase to the internalCa2+ store (Drenan et al., 2004). However, whether this is adirect effect of mTOR on the IP3R, or an indirect action, e.g.by cyclin-dependent protein kinase or PKC (Yonezawa et al.,2004; Malathi et al., 2003) remains to be established. mTORmight sense cellular ATP levels and suppress protein synthesiswhen these levels decrease (reviewed by Proud, 2002;

Houghton and Huang, 2004; Jaeschke et al., 2004), i.e. mTORmight act as a nutrient sensor for the cell. Rapamycin, byinhibiting mTOR, will mimic the conditions of nutritionaldepletion. Although the full functional significance of thepotentiating effect of mTOR on IP3R channel activity is stillunknown, it is tempting to suggest from our present findingsthat mTOR maintains Ca2+ release from IP3Rs when thenutritional status of the cell is adequate, whereas innutritionally-depleted conditions, IP3-mediated Ca2+ release isreduced to conserve ATP use like, for example, followinginhibition of Ca2+ pump activity in store refilling.

Different effects of rapamycin and FK506 on cell signallinghighlighted in this study are already recognised in othercontexts. For example, rapamycin inhibits smooth muscleproliferation, whereas FK506 does not (Poon et al., 1996).Here, we propose another example of different, indeedopposite, effects of FKBPs as revealed by FK506 andrapamycin on signals derived from IP3R. The diversity of rolesplayed by FKBP12 is achieved in part because FKBP12 has nodirect effect on the activity of the IP3R but might either increaseor decrease Ca2+ release indirectly, depending on the effectorspresent (calcineurin or mTOR).

The Wellcome Trust (060094/Z/00/Z) and British Heart Foundation(PG/2001079; PG/02/161) funded this work. The authors thank J.W.Craig for his excellent technical assistance, Fujisawa Inc. for the giftof FK506, Novartis Pharma AG for the gift of RAD001, Tim Seidler(University of Göttingen, Germany) for recombinant FKBP12 andPamela Scot and Susan Chalmers for helpful discussions.

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