Activation of IP₃ receptors requires an endogenous 1-8-14 calmodulin-binding motif

Abstract

Binding of IP₃ (inositol 1,4,5-trisphosphate) to the IP₃-binding core (residues 224–604) of IP₃Rs (IP₃ receptors) initiates opening of these ubiquitous intracellular Ca²⁺ channels. The mechanisms are unresolved, but require conformational changes to pass through the suppressor domain (residues 1–223). A calmodulin-binding peptide derived from myosin light chain kinase uncouples these events. We identified a similar conserved 1-8-14 calmodulin-binding motif within the suppressor domain of IP₃R1 and, using peptides and mutagenesis, we demonstrate that it is essential for IP₃R activation, whether assessed by IP₃-evoked Ca²⁺ release or patch-clamp recoding of nuclear IP₃R. Mimetic peptides specifically inhibit activation of IP₃R by uncoupling the IP₃-binding core from the suppressor domain. Mutations of key hydrophobic residues within the endogenous 1-8-14 motif mimic the peptides. Our results show that an endogenous 1-8-14 motif mediates conformational changes that are essential for IP₃R activation. The inhibitory effects of calmodulin and related proteins may result from disruption of this essential interaction.

INTRODUCTION

Ca²⁺ channels allow most electrical and many chemical signals to be transduced into the changes in cytosolic Ca²⁺ concentration that regulate almost every aspect of cellular activity [1]. Most Ca²⁺ channels are also regulated by Ca²⁺, either directly or via CaM (calmodulin) [2]. This provides feedback regulation of Ca²⁺ signalling and it allows Ca²⁺ channels to evoke regenerative Ca²⁺ signals [3]. The latter are important because they underpin the versatility of Ca²⁺ as an intracellular messenger, permitting it to function either locally or globally [1].

Two major families of intracellular Ca²⁺ channels, IP₃Rs [IP₃ (inositol 1,4,5-trisphosphate) receptors] and RyRs (ryanodine receptors), share many structural [4,5] and functional [5–7] properties. Most notably, all IP₃Rs and RyRs are stimulated by low concentrations of cytosolic Ca²⁺ and inhibited by higher concentrations. Ca²⁺-binding sites within the RyR itself can mediate this biphasic Ca²⁺ regulation [7], but, for IP₃Rs, it remains unclear whether additional Ca²⁺-binding proteins are required [6]. None of the many Ca²⁺-binding sites in RyRs [8] or IP₃Rs [9] has been unambiguously associated with Ca²⁺ regulation of channel gating [10,11], although mutation of a single equivalent residue in RyRs or IP₃Rs (Glu²¹⁰⁰ in IP₃R1) modulates their Ca²⁺-sensitivity [11]. Both families of intracellular Ca²⁺ channels are also regulated by CaM, a ubiquitously expressed and highly conserved Ca²⁺-binding protein [12]. Related proteins with EF-hand Ca²⁺-binding structures, such as S100A and CaBP1 (Ca²⁺-binding protein 1), also regulate RyRs and IP₃Rs, but the physiological significance of these interactions between intracellular Ca²⁺ channels and CaM or related proteins is unresolved [13,14]. Despite some conflicting evidence [15], CaM seems not to be essential for Ca²⁺ regulation of RyRs or IP₃Rs [16–18], but it does regulate both channels and it modulates their responses to Ca²⁺ [19–21].

All IP₃Rs are inhibited by Ca²⁺–CaM [22], but neither of the two CaM-binding sites within IP₃R1, nor a third that is created by alternative splicing [23], clearly mediates this inhibition of IP₃-evoked Ca²⁺ release. The central site [24] (Figure 1A) mediates neither Ca²⁺ nor CaM regulation of IP₃R activity [16,17] and it is absent from IP₃R3. The functional role of the split N-terminal site (Figure 1A), one component of which may also bind CaBP1 [25], is also unclear. It has been proposed to bind CaM and thereby to inhibit IP₃R activity, but only when Ca²⁺ has bound elsewhere [26]. The evidence that CaM inhibits IP₃R only in the presence of Ca²⁺, without CaM itself providing the Ca²⁺-sensor, is persuasive [26], but there is no compelling evidence to link this to the N-terminal CaM-binding site [27].

Figure 1. A putative 1-8-14 motif within the SD of the IP₃R.

(A) Key features of a rat IP₃R showing the NT, with its component parts (SD and IBC), the six C-terminal transmembrane domains (TMD) that form the pore and the CaM-binding domains (yellow). Residue numbers are shown. (B) Comparison of 1-8-14 motifs showing the conserved hydrophobic residues of the consensus sequence in blue. Charged residues within the 1-8-14 motif are highlighted in red because the consensus motif has a net charge of +3 to +6. The lower panel shows the peptides used with mutated residues underlined. (C) Structure of the SD of IP₃R1 (PDB code 1XZZ) and the equivalent region (A domain) of RyR1 (PDB code 3HSM) with the pseudo-1-8-14 motif highlighted and compared with MLCK in the structure it adopts when bound to Ca²⁺–CaM (PDB code 1QTX).

The links between CaM binding and function are better understood for RyRs, although the effects differ between RyR subtypes [7]. A single site on each RyR1 subunit (residues 3614–3643 in rabbit RyR1), which is conserved in all RyRs, binds the C-terminal lobe of both apo-CaM and Ca²⁺–CaM and appears to mediate the functional effects of CaM [20,28,29]. As this tethered CaM binds Ca²⁺, it migrates towards the NT (N-terminus) of the binding site and the CaM switches from activating RyR1 to inhibiting it [19]. The CaM-binding site of RyR1 also engages other CaM-like domains, notably the C-terminus of the L-type Ca²⁺ channel which inhibits RyR1 activity [30], and perhaps an EF-hand-like structure within the C-terminal region of RyR1 which binds Ca²⁺ and modulates Ca²⁺ regulation of RyR [31]. These observations suggest that the CaM-binding domain of RyR also mediates important inter- and intra-molecular interactions, and that the complex effects of CaM and related proteins may, at least in part, result from disrupting these interactions [29,31,32].

For IP₃Rs, IP₃ binding to the IBC (IP₃-binding core) (residues 224–604) (Figure 1A) initiates the conformational changes that lead to opening of a pore formed by the C-terminal transmembrane domains of each of the four IP₃R subunits [5,33]. These conformational changes pass via the N-terminal SD (suppressor domain) (residues 1–223), which is essential for IP₃R activation. Indeed, the major conformational changes associated with IP₃R activation appear to occur within the NT (residues 1–604) [5,33]. Although both IP₃ and Ca²⁺ are required for IP₃R activation [6,34], it is not yet clear how the conformational changes initiated by IP₃ lead to Ca²⁺ binding and then to gating of the pore. It is therefore intriguing that a CaM-binding peptide derived from MLCK (myosin light chain kinase), which comprises a 1-8-14 CaM-binding sequence [35], reversibly inhibits IP₃-evoked Ca²⁺ release [36] via all three vertebrate IP₃R subtypes. Furthermore, MLCK peptide is more potent in the presence of Ca²⁺ [35]. This inhibition is entirely independent of CaM and involves interaction of MLCK peptide with the NT in a manner that requires the SD [35]. We speculate, by analogy with RyRs, that inhibition of IP₃Rs by MLCK peptide might result from disruption of an interaction between endogenous CaM-like and CaM-binding domains within IP₃Rs, and that, for IP₃Rs, this interaction is essential for activation. In the present study, we explored this hypothesis further.

EXPERIMENTAL

Materials

Cell culture materials were from Gibco, except for fetal bovine serum (Sigma). CaM purified from bovine brain was from Calbiochem. [³H]IP₃ (18 Ci/mmol) was from PerkinElmer. IP₃ was from Alexis Biochemicals. Peptides were synthesized and purified by Sigma or New England Peptide, and each was shown to be >90% pure by HPLC. The peptide sequences are listed in Supplementary Table S1 (at http://www.BiochemJ.org/bj/449/bj4490039add.htm).

Site-directed mutagenesis

The NT (residues 1–604) and IBC (residues 224–604) of rat IP₃R1 were amplified by PCR from the full-length receptor clone lacking the SI splice region (GenBank® accession number GQ233032.1) as described previously [33]. The fragments were ligated into pTrcHis A (Invitrogen) to allow expression of N-terminally His₆-tagged proteins. Mutagenesis of the 1-8-14 motif within the NT used the QuikChange® II XL site-directed mutagenesis kit (Stratagene) for single mutants (F53E, L60E, Y66E and K52E) and the QuikChange® multi-site-directed mutagenesis kit for the double mutant (F53E and Y66E). The primers used are listed in Supplementary Table S2 (at http://www.BiochemJ.org/bj/449/bj4490039add.htm). The same primers and conditions were used for mutagenesis of full-length IP₃R using IP₃R1 in the pENTR 1A vector. Full-length constructs were subcloned into pcDNA3.2/V5-DEST for expression in DT40 cells. The complete sequence of every mutant construct was verified by sequencing.

Culture and stable transfection of DT40 cells

DT40 cells in which the genes for all three IP₃R subtypes had been disrupted (DT40-KO) [37] and DT40 cells stably expressing rat IP₃R1 (DT40-IP₃R1) were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) heat-inactivated chicken serum, 2 mM l-glutamine and 50 μM 2-mercaptoethanol. Cells were grown in suspension in 175 cm² flasks at 37°C in an atmosphere of 5% CO₂. They were used or passaged when they reached a density of ~2×10⁶ cells/ml. To generate stable cell lines expressing mutant IP₃R, the mutant construct in pcDNA3.2/V5-DEST was linearized, and DT40 cells were transfected by nucleofection (Amaxa, protocol B-23). Cell lines were selected with G-418 (2 mg/ml) and screened initially by Western blotting using a peptide antiserum to IP₃R1 [38] as described previously [33], and then using the functional assay described below.

Ca²⁺ release from the intracellular stores of permeabilized cells

The free Ca²⁺ concentration of the intracellular stores of permeabilized cells was measured using a low-affinity Ca²⁺ indicator trapped within the endoplasmic reticulum as reported previously [39]. Briefly, DT40 cells (4×10⁷ cells/ml) were suspended in HBS (Hepes-buffered saline: 135 mM NaCl, 5.9 mM KCl, 11.6 mM Hepes, 1.5 mM CaCl₂, 11.5 mM glucose and 1.2 mM MgCl₂, pH 7.3) containing 1 mg/ml BSA, 0.4 mg/ml Pluronic F127 and 20 μM mag-fluo-4/AM (Invitrogen). After 1 h at 20°C in the dark with gentle shaking, cells were centrifuged at 650 g for 2 min and resuspended to 10⁷ cells/ml in Ca²⁺-free CLM (cytosol-like medium) (20 mM NaCl, 140 mM KCl, 1 mM EGTA, 20 mM Pipes and 2 mM MgCl₂, pH 7.0) containing 20 μg/ml saponin. After incubation at 37°C with gentle shaking for 4 min, permeabilized cells were centrifuged at 650 g for 2 min and resuspended in Mg²⁺-free CLM, supplemented with CaCl₂ to give a final free Ca²⁺ concentration of 220 nM. The free Ca²⁺ concentration of CLM was calculated using the MaxChelator program (http://maxchelator.stanford.edu) and then measured using fluo-3 or fura-2. Cells were then washed, resuspended in Mg²⁺-free CLM containing 10 μM FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) to inhibit mitochondria, and distributed into a 96-well plate (10⁶ cells in 50 μl of CLM/well). After centrifugation, fluorescence from the luminal indicator was recorded using a FlexStation II platereader (Molecular Devices) equipped to allow automated additions [39]. In all experiments, the intracellular stores were allowed to load to steady-state with Ca²⁺ after addition of MgATP. IP₃ was then added with thapsigargin (1 μM, to inhibit Ca²⁺ reuptake). The Ca²⁺ release evoked by IP₃ is expressed as a fraction of the ATP-dependent Ca²⁺ uptake.

Patch-clamp recording

Currents were recorded from patches excised from the outer nuclear envelope of DT40 cells expressing recombinant rat IP₃R1 using symmetrical caesium methanesulfonate (140 mM) as the charge-carrier. The composition of recording solutions and methods of analysis were otherwise as described previously [40].

Expression of N-terminal fragments of IP₃R

The pTrcHis constructs were used for expression of N-terminally His₆-tagged proteins in Escherichia coli strain BL21(DE3) cells. Before use for [³H]IP₃ binding, proteins were cleaved from the His₆ tags using biotinylated thrombin (Novagen) at the engineered thrombin-cleavage site [33]. Complete cleavage was verified by Western blotting using an anti-His₆ antibody. The proteins were used for [³H]IP₃ binding without further purification [33].

[³H]IP₃ binding

Equilibrium-competition binding assays were performed at 4°C for 5 min in CLM (500 μl) with a free Ca²⁺ concentration of 220 nM and containing [³H]IP₃ (0.75–1.5 nM), bacterial lysate (10 μg of protein for IBC and 100 μg of protein for NT) or cerebellar membranes (50 μg of protein) and competing ligands. Non-specific binding was defined by addition of 10 μM IP₃. Bound and free [³H]IP₃ were separated by centrifugation at 20000 g for 5 min, after addition of poly(ethylene glycol) (15% final concentration) and γ-globulin (0.75 mg) for soluble proteins. Results were analysed by fitting to a Hill equation (using GraphPad Prism) from which the IC₅₀ (half-maximal inhibitory concentration) and thereby the K_d (equilibrium dissociation constant) were calculated [33].

Western blotting

Cells in Ca²⁺-free CLM containing 2-mercaptoethanol (1 mM) and protease inhibitors were lysed by addition of PopCulture (10%), lysozyme (10 μg/ml), DNAse (5 units/ml) and RNAse (10 μg/ml). The proteins were separated using SDS/PAGE pre-cast mini-gels (Invitrogen) and transferred on to a PVDF membrane using an Iblot dry-transfer apparatus (Invitrogen). The primary antibodies were rabbit anti-His₆ (1:3000 dilution) (Sigma) and anti-IP₃R1 (1:1000 dilution) [33]. HRP (horseradish peroxidase)-conjugated anti-rabbit secondary antibodies (1:5000 dilution) (AbCam) and the Super Signal West Pico chemiluminescence reagent (Pierce) were used to detect immunoreactivity. Bands were quantified using GeneTools software (Syngene).

Statistical analysis

For comparisons of K_d, EC₅₀ (half-maximally effective concentration) or IC₅₀ values, their negative logarithms (pK_d, pEC₅₀ and pIC₅₀; means±S.E.M.) were used for statistical analyses. For clarity, some Figures show normalized results, but all statistical analyses were performed on the raw data using paired or unpaired Student's t tests. P<0.05 was considered significant.

RESULTS AND DISCUSSION

Reversible inhibition of IP₃-evoked Ca²⁺ release by an endogenous 1-8-14 peptide

A sequence within the SD of all known IP₃Rs (residues 53–66 in rat IP₃R1; Supplementary Figure S1 at http://www.BiochemJ.org/bj/449/bj4490039add.htm) includes the critical hydrophobic residues of a 1-8-14 CaM-binding motif appropriately oriented within the known structure of the SD [41] (Figures 1B and 1C) and with the required net positive charge [35]. The sequence lies within one of the two regions (residues 49–81; Figure 1A) within the NT reported to bind CaM [42] and CaBP1 [14]. A similar sequence is present within the N-terminal of all RyRs (Supplementary Figure S1). To test our hypothesis that inhibition of IP₃R by MLCK peptide results from disruption of an essential interaction involving an endogenous 1-8-14 motif, we assessed the effects of a peptide derived from this motif (1-8-14 peptide; Figure 1B and Supplementary Table S1) on IP₃-evoked Ca²⁺ release.

The 1-8-14 peptide inhibited IP₃-evoked Ca²⁺ release via IP₃R1 without affecting either Ca²⁺ uptake or the sensitivity (EC₅₀) to IP₃ (Figures 2A–2D). A maximally effective concentration of the peptide reduced the maximal response to IP₃ by 77±7%. The IC₅₀ for 1-8-14 peptide was 767 μM (pIC₅₀, 3.1±0.25) (Figure 2C). Neither a mutant 1-8-14 peptide, in which two critical hydrophobic residues are mutated (1-8-14^C, 3 mM) nor a scrambled peptide (1-8-14^S, 3 mM) had any effect on IP₃-evoked Ca²⁺ release (Figure 2C). Both MLCK peptide (isoelectric point, pI 14.0) and 1-8-14 peptide (pI 11.6) are very basic and might therefore have inhibited IP₃-evoked Ca²⁺ release by binding directly to IP₃. We demonstrated previously that this was not the case for MLCK peptide [35], and it is also unlikely for the 1-8-14 peptide. The 1-8-14 and 1-8-14^S peptides are equally basic, but only the former inhibited IP₃R; the percentage inhibition caused by 3 mM 1-8-14 peptide is similar for all IP₃ concentrations (~75%), and neither was the inhibition reduced by increasing the IP₃ concentration beyond that required to stimulate maximal Ca²⁺ release (Figure 2B). We conclude that 1-8-14 peptide inhibits IP₃-evoked Ca²⁺ release by binding to IP₃R.

Figure 2. Inhibition of IP₃R by 1-8-14 peptide.

(A) Typical recording of the free Ca²⁺ concentration within the endoplasmic reticulum of a population of permeabilized DT40-IP₃R1 cells showing Ca²⁺ uptake after addition of MgATP (1.5 mM), release of Ca²⁺ after addition of IP₃ (10 μM, with 1 μM thapsigargin to inhibit Ca²⁺ re-uptake) and inhibition of that release by 1-8-14 peptide (3 mM, present throughout as indicated, upper trace). Results are means±S.E.M. for three replicates from a single experiment. (B) Concentration-dependent release of intracellular Ca²⁺ stores by IP₃ alone or after pre-incubation for 2.5 min with 1-8-14 peptide (3 mM). Inhibition by 1-8-14 peptide at each IP₃ concentration is also shown (%). 1-8-14 peptide caused a significant decrease in the maximal response (P<0.001) without significantly changing the sensitivity to IP₃. (C and D) Permeabilized cells pre-incubated for 10–20 min with the indicated concentrations of peptide were stimulated with IP₃ (10 μM, in the continued presence of peptide). Results show the Ca²⁺ content of the stores before addition of IP₃, and the Ca²⁺ release evoked by IP₃. (E) Permeabilized cells were incubated alone or with 1-8-14 peptide (3 mM) for 10–20 min, washed and then resuspended in CLM. Ca²⁺ release by IP₃ (10 μM) was then measured after a further incubation for 10–20 min with the indicated concentrations of 1-8-14 peptide. The Ca²⁺ release evoked by IP₃ with and without peptide is shown for naive cells and after the pre-treatment with 3 mM peptide. The results establish that the effects of 1-8-14 peptide are fully reversible. (F) Permeabilized cells pre-incubated with or without 1-8-14 peptide (1 mM) for 10–20 min were stimulated with a maximally effective concentration of IP₃ in the continued presence of peptide in CLM with the indicated free Ca²⁺ concentration. Results show the inhibition of IP₃-evoked Ca²⁺ release (%) by 1-8-14 peptide at each free Ca²⁺ concentration. Results in (B)–(F) are means±S.E.M. (n≥3). *P<0.05, **P<0.01 and ***P<0.001.

The 1-8-14 peptide is only 16 residues long. A longer peptide (30 residues, 1-8-14^L), which includes additional N- and C-terminal residues that are conserved in all IP₃Rs (Figure 1B and Supplementary Figure S1), also inhibited IP₃-evoked Ca²⁺ release without affecting Ca²⁺ uptake (Figure 2D). Although IP₃R may be slightly more sensitive to the longer peptide (IC₅₀, 326 μM; pIC₅₀, 3.5±0.25) than to the 1-8-14 peptide (767 μM, 3.1±0.25); the difference was not statistically significant. Subsequent studies used the shorter 1-8-14 peptide because it was less expensive.

The results shown in Figure 2(E) demonstrate that the effects of a maximally effective concentration of 1-8-14 peptide (3 mM) are fully reversible. These experiments, which require extensive washing of the cells between successive challenges with the peptide, confirm that the inhibition of IP₃Rs by the 1-8-14 peptide, like that by MLCK peptide [35], does not result from dissociation of CaM from IP₃R [36]. Our previous study demonstrated that MLCK peptide more potently inhibited IP₃R when the cytosolic free Ca²⁺ concentration was increased [35]. Similar results were obtained with 1-8-14 peptide (Figure 2F). We conclude that 1-8-14 peptide inhibits IP₃-evoked Ca²⁺ release by binding to the IP₃R and the inhibition is enhanced at elevated cytosolic Ca²⁺ concentrations.

Inhibition of single-channel currents through IP₃Rs by 1-8-14 peptide

In patch-clamp recordings from the nuclear envelope of DT40 cells expressing rat IP₃R1, a maximally effective concentration of IP₃ stimulated IP₃R activity and this was massively attenuated by the 1-8-14 peptide (3 mM) (Figures 3A and 3B). Our results are consistent with the peptide causing a 50% decrease in the mean channel open time (τ_o) (Figure 3C). However, the overall channel activity (NP_o) was so low under these conditions that we cannot reliably estimate the number of active IP₃Rs (N) within each patch. We cannot therefore entirely eliminate the possibility that each patch fortuitously included several IP₃Rs and that their clustering caused τ_o to fall from ~10 ms to ~5 ms as we reported previously [40]. An effect on τ_o would be unusual because most regulators of IP₃Rs affect the duration of closed states (τ_c) [6,40]. The effect of the peptide on τ_o is not, however, sufficient to account for the ~10-fold decrease in NP_o (Figure 3A), suggesting that the 1-8-14 peptide must also affect the rate of channel opening (i.e. τ_c). Because it was impossible to determine the number of active IP₃Rs in the presence of 1-8-14 peptide (see above), we could not reliably determine τ_c. The single-channel conductance (γ_Cs) was unaffected by 1-8-14 peptide: it was 214±6 pS (n=3) and 209±6 pS (n=3) for control and peptide-treated IP₃Rs respectively (Figure 3D).

Figure 3. Inhibition of IP₃R gating by 1-8-14 peptide.

(A) Typical recordings from excised nuclear patches stimulated with IP₃ (10 μM) with and without 1-8-14 peptide (3 mM) in the pipette solution. The holding potential was +40 mV. The closed state is shown. (B and C) NP_o (B) and τ_o (C) for IP₃R stimulated with IP₃ alone or with 1-8-14 peptide (3 mM, +Pep). Results for IP₃R^L60E are also shown. *P<0.05, ***P<0.001 and ****P<0.0001 relative to native IP₃R without peptide. (D) Single-channel current (i)–voltage (V) relationships for the three stimulation conditions. Results in (B)–(D) are means±S.E.M. (n≥3).

These results establish that a peptide derived from an endogenous 1-8-14 motif within the SD of the IP₃R is similar to MLCK peptide in causing substantial and reversible inhibition of IP₃Rs that is independent of CaM. This conclusion is consistent with our suggestion that MLCK peptide inhibits IP₃Rs by mimicking an endogenous 1-8-14 motif, and so perhaps ‘unzipping’ an interdomain interaction [43] that is essential for activation of IP₃Rs.

1-8-14 peptide uncouples IP₃ binding from activation of IP₃Rs

Removal of the SD increases the affinity of both full-length IP₃Rs and the NT for IP₃ [33]. We [33] have suggested that this reflects the use of binding energy to drive conformational rearrangement of SD-IBC interfaces during the initial steps of IP₃R activation [5,44].

1-8-14 peptide (3 mM) increased specific binding of [³H]IP₃ to full-length IP₃R1. Similar results were obtained with the NT, but IP₃ binding to the IBC was unaffected (Figure 4A). The latter demonstrates that 1-8-14 peptide does not interact directly with either the IP₃-binding site or with IP₃. Neither the mutated (1-8-14^C) nor scrambled (1-8-14^S) peptide had any effect on IP₃ binding to the NT (Figure 4A). These results with IP₃R fragments expressed in E. coli, which lack CaM, also further support our conclusion that the effects of 1-8-14 peptide are entirely independent of CaM.

Figure 4. 1-8-14 peptide directly stimulates IP₃ binding to the NT of IP₃R.

(A) Specific equilibrium binding of [³H]IP₃ (1.5 nM) to membranes from rat cerebellum (full-length IP₃R, FL) or to isolated NT or IBC, alone or in the presence of 3 mM of the indicated peptide. *P<0.05 and **P<0.01 relative to control; comparisons were performed on the raw data. (B) Concentration-dependent effects of 1-8-14 peptide on specific [³H]IP₃ binding to NT in CLM with 220 nM free Ca²⁺ concentration, plotted as the increase in specific [³H]IP₃ binding as a percentage of that evoked by the maximal concentration of peptide. Results are means±S.E.M. (n≥3).

Comparison of the effects of 1-8-14 peptide on stimulating [³H]IP₃ binding to the NT (EC₅₀, 615 μM; pEC₅₀, 3.21±0.19) (Figure 4B) with its inhibitory effect on IP₃-evoked Ca²⁺ release (IC₅₀, 767 μM; pIC₅₀, 3.1±0.25) (Figure 2C) demonstrates that each is similarly sensitive to the peptide. These results are consistent with our hypothesis that the 1-8-14 peptide disrupts an interaction between the SD and IBC that is essential for IP₃R activation. The peptide thereby inhibits IP₃-evoked Ca²⁺ release (Figure 2) and IP₃R activity (Figure 3) and, by uncoupling IP₃ binding from subsequent conformational changes, it stimulates IP₃ binding (Figure 4). Subsequent experiments used mutagenesis of residues within the endogenous 1-8-14 motif to test this hypothesis further.

Mutations within the endogenous 1-8-14 sequence increase IP₃-binding affinity

If, as we suggest, the 1-8-14 peptide disrupts an essential interaction between the endogenous 1-8-14 sequence and another domain within the NT, we might expect mutation of appropriate residues in the SD to both disrupt IP₃R activation and increase IP₃-binding affinity. We tested the latter prediction by examining IP₃ binding to the NT in which each of the critical (1, 8 and 14) hydrophobic/aromatic residues that are important for Ca²⁺–CaM binding to 1-8-14 motifs [45] was replaced with a charged hydrophilic residue (glutamate). The same hydrophobic residues are essential for MLCK [35] and 1-8-14 (Figure 2C) peptides to disrupt IP₃R activation.

NTs of IP₃R1 with point mutations in positions equivalent to the 1- (F53E), 8- (L60E) or 14-position (Y66E) of the endogenous 1-8-14 motif (Figure 1A) were expressed in E. coli. Expression levels of the NT and its mutants were not identical (Figure 5A), but they were each sufficient to allow the affinity for IP₃ and the effects of peptides to be determined after cleavage of the His₆ tag, but without further purification [33]. As expected, IP₃ bound to the IBC with greater affinity (17-fold) than to the NT (Figure 5B) [33,46,47], consistent with our suggestion that, in the absence of the SD, less binding energy is diverted into conformational changes [33]. Mutation of critical residues within the endogenous 1-8-14 motif significantly increased the affinity of the NT for IP₃ (Figure 5B and Table 1), although none was as effective as complete removal of the SD. This is consistent with our observation that neither the 1-8-14 (Figure 2) nor MLCK [35] peptide entirely inhibits IP₃-evoked Ca²⁺ release, whereas removal of the SD totally uncouples IP₃ binding from IP₃R activation [48]. Although maximally effective concentrations of MLCK (100 μM) or 1-8-14 (3 mM) peptides similarly increased IP₃ binding to the NT, neither peptide had any effect on [³H]IP₃ binding to the NT with mutations in any of the critical 1-8-14 residues (Figures 5C and 5D). Mutation of a residue immediately preceding the critical 1-position of the 1-8-14 motif (K52E), which did not increase the affinity of IP₃ for the NT (Supplementary Figure S2A at http://www.BiochemJ.org/bj/449/bj4490039add.htm), had no effect on the responses to MLCK or 1-8-14 peptides (Figures 5C and 5D) and neither did it affect IP₃-evoked Ca²⁺ release [33] (Supplementary Figure S2B). These results establish that mutation of critical residues within the endogenous 1-8-14 motif selectively increases IP₃-binding affinity and these effects are non-additive with those of either MLCK or 1-8-14 peptide.

Figure 5. Mutations within the 1-8-14 motif mimic the effect of 1-8-14 peptide on IP₃ binding.

(A) Western blot (typical of three independent experiments) with an anti-His₆ antibody of lysates (5 μg of protein/lane) from bacteria expressing NT with the indicated mutations. The 80 kDa molecular-mass marker is shown. (B) Concentration-dependent effect of IP₃ on specific [³H]IP₃ binding to the IBC, NT and mutated NT. (C and D) Effects of MLCK peptide (C, 100 μM) and 1-8-14 peptide (D, 3 mM) on specific binding of [³H]IP₃ (1.5 nM) to the NT and the indicated mutants (each expressed as a percentage of the control). (E) Specific binding of [³H]IP₃ (1.5 nM) to the IBC, NT and mutated NT in the presence of the indicated concentrations of heparin. (F) Summary results from experiments similar to those in (E) showing the K_d for IP₃ and heparin binding to the IBC, NT and mutated NT. Results in (B)–(F) are means±S.E.M. (n≥3). *P<0.05, **P<0.01 and ***P<0.001 relative to control; comparisons were performed on the raw data.

Table 1. Binding of IP₃ and heparin to N-terminal fragments of IP₃R1.

Equilibrium competition binding using [³H]IP₃ was used to measure the pK_d of IP₃ and heparin for the N-terminal fragments of IP₃R1. Affinities for ligands are also shown expressed as fold increase relative to wild-type NT (i.e. K_d^NT/K_d^mutant). Results are means±S.E.M. (n≥3). *P<0.05, **P<0.01 and ***P<0.001 relative to NT.

Fragment	pKd, /M IP3 (Kd, nM)	Affinity relative to NT	pKd, /g/ml heparin (Kd, ng/ml)	Affinity relative to NT
NT	7.40±0.11 (40.0)	1	6.62±0.06 (239)	1
F53E	7.97±0.05** (10.8)	4	6.92±0.06** (120)	2
L60E	7.84±0.08* (14.5)	3	6.70±0.04 (200)	1.2
Y66E	7.64±0.04 (22.8)	2	6.77±0.03 (171)	1.4
IBC	8.62±0.05*** (2.4)	17	6.93±0.04** (117)	2.0

Mutations within the 1-8-14 motif selectively increase agonist affinity

Our hypothesis is that the apparent affinity of agonists (such as IP₃) for native IP₃Rs is reduced because some of their binding energy is diverted into the conformational changes that activate the IP₃R [33]. Antagonists, because they need not evoke the rearrangement of the IBC and SD that initiates IP₃R activation, may be less affected by disruption of these interactions. We therefore examined the effects of the SD and of point mutations within the endogenous 1-8-14 sequence on binding to the NT of heparin, a competitive antagonist of IP₃ [49]. The results demonstrate that, whereas removal of the SD increased the affinity of the NT for IP₃ 17-fold, it caused only a 2-fold increase in the affinity for heparin. Point mutations within the endogenous 1-8-14 motif also caused larger increases in the affinity for IP₃ than for heparin (Figures 5E and 5F, and Table 1). These results are important because they demonstrate that the effects of the SD and of mutations within the 1-8-14 sequence on ligand binding are specific for an agonist of the IP₃R. They thereby demonstrate the importance of the 1-8-14 motif in specifically mediating activation of IP₃Rs.

Mutations within the endogenous 1-8-14 motif uncouple IP₃ binding from gating of IP₃Rs

It proved difficult to establish stable DT40 cell lines expressing rat IP₃R1 in which critical residues within the 1-8-14 motif were mutated, but we succeeded with two mutants (Figure 6A). The first (IP₃R^L60E) is mutated at the 8-position of the 1-8-14 motif and the second has mutations at both the 1- (F53E) and 14-positions (Y66E) (IP₃R^FY). As expected, a maximally effective concentration of IP₃ (10 μM) failed to stimulate Ca²⁺ release from permeabilized DT40 cells lacking IP₃R (DT40-KO cells) [37,40], but it caused release of 81±1% of the Ca²⁺ stores of DT40-IP₃R1 cells (Figures 6B and 6C). In the cell lines expressing IP₃R with a mutated 1-8-14 motif, there was barely detectable Ca²⁺ release that was not significantly different from that observed in DT40-KO cells (Figures 6B and 6C). ATP-dependent Ca²⁺ uptake into the ER was similar for each cell line (Figure 6C). We were concerned that the lower level of expression of mutant IP₃R relative to wild-type (~30–50%, Figure 6A) might have contributed to the lack of detectable IP₃-evoked Ca²⁺ release. However, in another stable DT40 cell line where the IP₃-binding site was mutated (R568Q), causing a ~10-fold decrease in IP₃ affinity [50], IP₃R expression (~15% of wild-type) was less than half that of the cell lines with mutations in the 1-8-14 motif (Figure 6A). Nevertheless, IP₃ caused a readily detectable release of Ca²⁺ from the intracellular stores of DT40-IP₃R^R568Q cells (49±2% of that detected in DT40-IP₃R1 cells) (Figures 6B and 6C). We conclude that the lack of detectable Ca²⁺ release in cells expressing IP₃R with a mutant 1-8-14 motif is not attributable to reduced IP₃R expression. Neither is it likely that the lack of response to IP₃ from mutant IP₃R reflects a more global disruption of IP₃R structure because each of the full-length mutant IP₃Rs bound IP₃, although, as predicted, addition of MLCK peptide increased IP₃ binding to only the wild-type IP₃R (Figure 6D). Furthermore, DT40 cells expressing IP₃R1 with a mutation in an adjacent residue (DT40-IP₃R1^K52E) responded normally to IP₃ [33] (Supplementary Figure S2B). These results are consistent with the suggestion that mutations within the endogenous 1-8-14 motif mimic addition of exogenous MLCK peptide by uncoupling IP₃ binding from the conformational changes that lead to opening of the IP₃R pore. Single-channel analyses provide further support for this conclusion.

Figure 6. The endogenous 1-8-14 motif is essential for activation of IP₃R.

(A) Expression of IP₃R1 in DT40 cells stably expressing each of the indicated mutants. Each lane was loaded with 4×10³ cells and probed with antisera to IP₃R1 (upper panel) or β-adaptin (lower panel). The R568Q mutant (which reduces the affinity of the IP₃R for IP₃) [50] is shown because it provides a control for functional assays of cells expressing IP₃R at low density. Molecular-mass markers are shown on the right. The Western blot is typical of three independent experiments. The lower panel shows summary results (means±S.E.M., n=3), where IP₃R expression was calculated from blots that included DT40-IP₃R1 membranes in which levels of expression were established by equilibrium competition [³H]IP₃ binding. (B) Typical responses to IP₃ (10 μM) from DT40 cells lacking IP₃R (KO) or expressing wild-type IP₃R1 or IP₃R with the indicated mutations (see the text for details). (C) Summary results show the Ca²⁺ content of the loaded stores (●) and the Ca²⁺ released by IP₃ (histograms) for each of the indicated cell lines. (D) Specific [³H]IP₃ binding (1.5 nM) to full-length IP₃R (FL) with the indicated mutations (L60E or FY, see the text for details) in permeabilized DT40 cells alone or in the presence of 100 μM MLCK peptide. Results in (C) and (D) are means±S.E.M. (n≥3). (E) Typical records from active excised nuclear patches of DT40 cells expressing IP₃R1 or IP₃R1^L60E stimulated with IP₃ (10 μM). The holding potential was +40 mV. C denotes the closed state. Summary data are provided in Figures 3(B)–3(D). **P<0.01, ***P<0.001 and ****P<0.0001 relative to IP₃R1 (A and C) or control (D).

Yamazaki et al. [51] reported recently the functional effects of mutations within IP₃R including some within the 1-8-14 motif (F53D and Y66A). We note, however, that some of their mutations, e.g. Y167A, which is clearly implicated in IP₃R activation, abolished IP₃-evoked Ca²⁺ release from microsomes without affecting Ca²⁺ signals evoked by activation of the BCR (B-cell receptor) in intact cells. This unexplained disparity casts some doubt over whether in these assays responses from intact cells faithfully report the activity of IP₃R. In DT40 cells expressing an IP₃R with five mutations that included Y66A (the 14-position of the 1-8-14 motif), activation of the BCR evoked a Ca²⁺ signal, suggesting that the mutant IP₃R was functional [51]. However, in this IP₃R, the mutant had one hydrophobic residue replaced by another and this might not radically affect the behaviour of the 1-8-14 motif. In preliminary analyses of cells expressing IP₃Rs in which the first position of the 1-8-14 motif was mutated (F53D), Ca²⁺ signals were also observed after activation of the BCR [51]. This may reflect a limitation of the BCR-based assay (see above) or it may provide evidence for a lesser role of the 1-position in the 1-8-14 motif. We have not succeeded in establishing a DT40 cell line expressing IP₃Rs with only this mutation, although our results do clearly show that IP₃Rs with mutations in both the 1- and 14-positions (IP₃R^FY) are barely responsive to IP₃ (Figure 6).

Mutation of the endogenous 1-8-14 motif attenuates IP₃R gating without affecting single-channel conductance

In keeping with the reduced expression of IP₃R^L60E in DT40 cells (Figure 6A), the frequency with which functional IP₃Rs were detected in excised nuclear patches was much lower for nuclei from paired experiments with DT40-IP₃R1^L60E cells (three of 48 patches) than from DT40-IP₃R1 cells (five of 13 patches). In parallel analyses, functional IP₃Rs were never detected in DT40-KO cells (none of 30 patches). The single-channel conductances (γ_Cs) of the mutant IP₃R^L60E (209±8 pS) and normal IP₃R (214±6 pS) were indistinguishable (Figure 3D), but NP_o was massively decreased in the mutant (Figures 3B and 6E). Our interpretation of the latter is, as we described in our analyses of the 1-8-14 peptide, limited by our inability, when NP_o is so low for IP₃R1^L60E, to estimate reliably the number of active IP₃R within a patch. Nevertheless, it is clear that the major effect on single-channel behaviour of mutating the endogenous 1-8-14 motif of IP₃R1 (Figures 3B–3D and 6E) and of adding 1-8-14 peptide to normal IP₃R1 (Figure 3) is similar: both decrease NP_o without affecting γ_Cs. These results establish that mutations in the endogenous 1-8-14 motif or addition of 1-8-14 peptide uncouple ligand binding from channel gating without compromising the behaviour of the pore.

Conclusions: interactions between endogenous 1-8-14 and CaM-like motifs mediate activation of IP₃Rs

CaM [22] or related EF-hand-containing proteins [14,25], peptides that comprise 1-8-14 CaM-binding motifs [35,36] (Figures 2–4) or disruption of a conserved endogenous 1-8-14-like motif within the SD of IP₃Rs inhibit IP₃-evoked Ca²⁺ release (Figures 5 and 6) by massively reducing NP_o of IP₃R (Figures 3 and 6E). We conclude that an endogenous 1-8-14 motif within the SD (Figure 1) is essential for IP₃R activation. Where it has been examined, the inhibitory proteins or peptides are more potent when Ca²⁺ is bound to the IP₃R [26,35] (Figure 2F). We therefore speculate that the endogenous 1-8-14 motif may interact with an unidentified domain that includes an EF-hand-like structure and that these interactions might be related to Ca²⁺ regulation of IP₃R (Figure 7). We suggest that competing peptides (CaM-like or 1-8-14 motifs) or mutagenesis of the endogenous 1-8-14 motif inhibit IP₃Rs by disrupting this essential interaction in a manner similar to the ‘unzipping’ of interdomain interactions in RyRs [32,43,52]. The scheme is appealing because IP₃ regulates binding of Ca²⁺ to IP₃Rs and thereby leads to channel gating [34,53]. The identity of this Ca²⁺-binding site is unknown. It is, however, clear that Ca²⁺ regulates IP₃ binding to the NT only when the SD is present [42], suggesting that a Ca²⁺-binding site within the NT may be regulated by interactions between the SD and IBC. One possibility is that an endogenous EF-hand-like structure might provide the Ca²⁺-binding site and that its interaction with the 1-8-14 motif links IP₃ and Ca²⁺ binding (Figure 7A). Bioinformatic analyses had suggested the presence of two possible EF-hand-like structures within the IBC [9,54], but neither is evident in high-resolution structures of the IBC [55] and NT [5,56]. Neither have we succeeded in identifying a complementary partner of the 1-8-14 motif. Another possibility is suggested by comparison of the structures of the NT with and without IP₃ bound [5,56], which reveal that Phe⁵³ (the first hydrophobic residue of the 1-8-14 motif) and Phe²²³ are closely apposed (~3.9 Å; 1 Å=0.1 nm), but they move apart (~5.3 Å) when IP₃ binds (Figure 7B). A β-sheet links Phe²²³ to Glu²⁴⁶, and the movement of Phe²²³ is associated with a repositioning of an acidic residue in the β-domain of the IBC (Glu²⁴⁶). This brings Glu²⁴⁶ closer to three other acidic residues (Glu⁴²⁵, Asp⁴²⁶ and Glu⁴²⁸). The rearrangement is interesting because these four residues have been proposed to form a Ca²⁺-binding site (Ca-I) [55]. Furthermore, a peptide (residues 378–450) that includes most of these residues binds Ca²⁺, and the binding is abolished by mutation of the acidic residues [42]. A second possibility is therefore that IP₃-evoked movement of the critical 1-8-14 motif contributes to formation of an effective Ca²⁺-binding site within the IBC by bringing a fourth acidic residue into appropriate association with three others.

Figure 7. Activation of IP₃Rs requires an endogenous 1-8-14 motif.

IP₃ binding to the IBC initiates conformational changes that pass via the SD and lead, via regulation of Ca²⁺ binding to the IP₃R, to opening of the pore [33]. (A) An endogenous 1-8-14 motif within the SD is essential for IP₃R activation. We speculate (upper panel) that interaction of this CaM-binding motif (red, conserved hydrophobic residues in dark blue) with an endogenous, but presently unknown, CaM-like structure (pale blue) within the NT may link IP₃ binding to Ca²⁺ binding. (B) Another possibility is that IP₃ binding rearranges the 1-8-14 motif and so repositions a critical acidic residue (Glu²⁴⁶) that may then contribute to a Ca²⁺-binding site (Ca-1) [55]. The NT without IP₃ bound (PDB code 3UJ0) [5] is shown with the IBC in grey and the SD in green to highlight Phe⁵³ (within the 1-8-14 motif) and Phe²²³ to which it is closely apposed (yellow box), residues proposed to form the Ca-1 site (pink box) and the β-sheet that links Phe²²³ to Glu²⁴⁶ (cyan box). The expanded views (each rotated to show key movements) show the critical residues and the linking β-sheet before (green) and after IP₃ binding (blue, PDB code 3UJ4). The carboxy oxygen atoms in Glu²⁴⁶ are shown in magenta. We speculate that separation of Phe⁵³ and Phe²²³ when IP₃ binds is associated with twisting of the linking β-sheet and movement of Glu²⁴⁶ towards three other acidic residues (Glu⁴²⁵, Asp⁴²⁶ and Glu⁴²⁸) and that they may then together form an effective Ca²⁺-binding site.

We conclude that a conserved 1-8-14 motif within the SD is essential for IP₃R activation and speculate that its interaction with either an endogenous CaM-like motif or acidic residues within the IBC may link IP₃ and Ca²⁺ binding. Inhibition of IP₃R by CaM and related proteins probably results from disruption of this essential interaction.

AUTHOR CONTRIBUTION

Yi Sun and Ana Rossi performed the Ca²⁺-release and IP₃-binding analyses. Taufiq Rahman performed the single-channel analyses. Colin Taylor directed the study, and with input from all authors, wrote the paper.

FUNDING

Supported by the Wellcome Trust [grant number 085295], Biotechnology and Biological Sciences Research Council [grant number BB/H009736/1] and a studentship from the Engineering and Physical Sciences Research Council (to Y.S.). A.R. is a fellow of Queens’ College, Cambridge. T.R. is a Drapers Research Fellow of Pembroke College, Cambridge.