Regulation of Inositol 1,4,5-Trisphosphate Receptors by cAMP Independent of cAMP-dependent Protein Kinase

Abstract

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP₃R) to IP₃ that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP₃R2. Using DT40 cells expressing single subtypes of mammalian IP₃R, we demonstrate that high concentrations of cAMP similarly sensitize all IP₃R isoforms to IP₃ by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP₃ binding to IP₃R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP₃. In single channel recordings from excised nuclear patches of cells expressing IP₃R2, cAMP alone had no effect, but it increased the open probability of IP₃R2 activated by a submaximal concentration of IP₃ alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP₃R subtypes to IP₃. For IP₃R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP₃. Using stably expressed short hairpin RNA to reduce expression of the G-protein, Gα_s, we demonstrate that attenuation of AC activity by loss of Gα_s more substantially reduces sensitization of IP₃R by PTH than does comparable direct inhibition of AC. This suggests that Gα_s may also specifically associate with each AC·IP₃R complex. We conclude that all three subtypes of IP₃R are regulated by cAMP independent of PKA. In HEK cells, where IP₃R2 selectively associates with AC6, Gα_s also associates with the AC·IP₃R signaling junction.

Introduction

Ca²⁺ and cAMP are two of a limited number of intracellular messengers used by cells to regulate a diverse array of cellular events in response to many different extracellular stimuli. The specificity of these messengers is provided by the spatio-temporal organization of their concentration changes within cells (1–4) and by complex interactions between them (2, 5, 6). In addressing the means whereby parathyroid hormone (PTH)³ regulates release of Ca²⁺ from intracellular stores, we have shown that PTH acts entirely via cAMP to increase the sensitivity of IP₃ receptors (IP₃R) to IP₃, thereby potentiating the Ca²⁺ signals evoked by other receptors that stimulate IP₃ formation (7). This effect of PTH is not mediated by the common targets of cAMP, PKA, and exchange proteins activated by cAMP (epac), but results instead from cAMP binding directly to a low affinity site on either the IP₃R itself or a protein tightly associated with it (7). These results identify the IP₃R as a new target for regulation by cAMP and so reveal another site at which cAMP and Ca²⁺ signaling pathways interact. The results also highlighted the importance of the precise spatial relationship between adenylyl cyclase (AC) and IP₃R, because cAMP appears to pass directly and selectively from type 6 AC (AC6) to the type 2 IP₃R (IP₃R2) via an association that we termed an AC·IP₃R junction (7) (Fig. 1A).

FIGURE 1.

PTH potentiates CCh-evoked Ca²⁺ release via a direct effect of cAMP on IP₃R. A, binary and analog modes of cAMP signaling (7). AKAP, protein kinase A-anchoring protein; PM, plasma membrane. Further details are in text. B, populations of HEK-PR1 cells in Ca²⁺-free HBS were stimulated with CCh alone (1 mm, black line) or with PTH (100 nm, 60 s) followed by CCh (gray line). Results are typical of at least three similar experiments. C, HEK-PR1 cells were preincubated (20 min) with the indicated concentrations of 8-Br-cAMP alone or with H89 (10 μm) (7) before stimulation with a submaximal concentration of CCh (1 μm). Results show the increase in [Ca²⁺]_i evoked by the CCh addition. D, IP₃-evoked Ca²⁺ release from permeabilized HEK-PR1 cells in the presence of submaximal (1 mm) or maximal (10 mm) concentrations of cAMP after preincubation with or without H89 (10 μm; 20 min). Results (C and D) show means ± S.E., n = 3 independent experiments.

These observations led us to define two forms of local cAMP signaling (Fig. 1A). The first mode, represented by the AC·IP₃R junction, is binary signaling. Here, cAMP is delivered directly to its target at very high concentrations by a closely associated AC; cAMP then switches the target on in all-or-nothing fashion. The second mode is analog signaling, where targets further away from AC, like PKA and exchange proteins activated by cAMP, respond to graded changes in cAMP concentration. The low affinity of the IP₃R for cAMP ensures that diffusion of cAMP is probably sufficient to terminate a response and insulate one junction from its neighbors. For analog signaling, local degradation of cAMP by associated cyclic nucleotide phosphodiesterases is required to terminate the response and maintain the spatial organization of cAMP signaling (Fig. 1A) (7, 8).

Here, we address two further questions related to cAMP signaling to IP₃R. First, although we have shown that, in intact HEK cells the effects of PTH are selectively mediated by IP₃R2, it is unclear whether this results solely from a selective association of IP₃R2 with AC6, or whether IP₃R2 is also unique among the three IP₃R subtypes in responding to cAMP. We demonstrate, using IP₃R1–3 expressed in DT40 cells lacking native IP₃R, that all IP₃R subtypes are regulated by cAMP itself. Second, although AC6 and IP₃R2 are associated in a signaling complex (the AC6·IP₃R2 junction, Fig. 1A), it is unclear whether other components of the signaling pathway linking PTH receptors to sensitization of IP₃R are also uniquely associated with individual AC6·IP₃R junctions. By selectively inhibiting Gα_s expression using stably expressed shRNA, we demonstrate that Gα_s is probably also specifically associated with AC·IP₃R junctions.

EXPERIMENTAL PROCEDURES

Cells and Vectors

HEK 293 cells stably transfected with human type 1 PTH receptor (HEK-PR1 cells) were cultured as described (9). A modified pSUPER vector (10) was used for shRNA-mediated knockdown of Gα_s in HEK-PR1 cells. An expression cassette for blasticidin resistance from pcDNA6/TR (Invitrogen) was cloned upstream of the polymerase-III H1-RNA promoter of pSUPER to generate a pSUPERBlank vector. Primers 1 and 2 for human Gα_s (see supplemental Table S1 for primer sequences) were annealed, phosphorylated by T4 polynucleotide kinase (New England Biolabs), and ligated into the BglII-HindIII sites of pSUPERBlank to create the pSUPERGα_s vector. The plasmids, pSUPERBlank and pSUPERGα_s, were linearized and transfected into HEK-PR1 cells (5 μg of DNA/10⁶ cells) using Transfast^TM Reagent (Promega). Transfected cells were selected using blasticidin (10 μg/ml) and G418 (800 μg/ml). Clonally isolated cells were screened for Gα_s by immunoblotting using a Gα_s antiserum (RM/1, 1:1000, New England Nuclear). Appropriate clones were further propagated for use in assays of cAMP and [Ca²⁺]_i.

DT40 cells in which each IP₃R gene had been disrupted (DT40KO cells) (11) were cultured as described (12). The ORF of rat IP₃R1 (GenBank^TM accession number GQ233032.1) was amplified by PCR from the expression vector pCMVI-9-IP₃R1 (13) using primers 3 and 4 (supplemental Table S1), and cloned as an EcoRI fragment into pENTR1a vector (Invitrogen). The ORF of mouse IP₃R2 (GenBank^TM accession number AB182290) was amplified by PCR as two fragments from the expression vector pcDNA3-IP₃R2 (14). The 5′ fragment (1–3297) was amplified by PCR using primers 5 and 6 and cloned as a SalI-XhoI fragment into pENTR1a. Then the entire ORF (1–8196) was amplified by PCR using primers 5 and 7, digested with KpnI and XhoI, and the resulting fragment (2230–8196) was cloned into pENTR1a. Finally, a SalI-BstBI fragment (1–2711) from the first construct was cloned into the second construct (replacing the smaller SalI-BstBI fragment) to give the entire ORF (1–8196) of mouse IP₃R2 in pENTR1a. Supplemental Experimental Procedures provide further details of the cloning and expression of IP₃R2. The ORF of rat IP₃R3 (GenBank^TM accession number GQ233031.1) was amplified by PCR from the expression vector pCB6-IP₃R3 (15) using primers 8 and 9, and cloned as an EcoRI fragment into pENTR1a vector (Invitrogen). Each pENTR1a-IP₃R construct was recombined with pcDNA3.2/V5-DEST (Invitrogen) to generate the Gateway-compatible expression vectors pcDNA3.2-IP₃R1 (2 and 3). DT40KO cells were transfected by nucleofection with linearized constructs of pcDNA3.2-IP₃R1 (2 or 3) using solution T and program B23 (Amaxa) using 5 μg of DNA/10⁶ cells. G418 (2 mg/ml) was used to select and amplify clones of cells. Expression of IP₃R in each cell line was quantified by immunoblotting using an anti-peptide antiserum (AbC (16)) that interacts equally with all three IP₃R subtypes. GeneTools (Syngene) was used to quantify band intensities on immunoblots. To estimate numbers of IP₃R in each DT40 cell line, immunoblots were calibrated using membranes prepared from rat cerebellum in which the receptor density (B_max) had been measured by equilibrium competition [³H]IP₃ binding.

For expression of mouse IP₃R2 in Sf9 cells, pENTR1a-IP₃R2 was recombined with Gateway-compatible BaculoDirect C-Term linear DNA (Invitrogen) to generate a recombinant baculovirus using the BaculoDirect^TM C-Term expression kit (Invitrogen). Sf9 cells were cultured as previously described (17), they were then infected with the recombinant virus DNA, and the virus was isolated according to the manufacturer's instructions (Invitrogen). For IP₃R2 expression, cells were infected at a multiplicity of infection of 10.

Measurements of IP₃-evoked Ca²⁺ Release from Permeabilized Cells

The intracellular stores of DT40 cells stably expressing mammalian IP₃R were loaded with Mag-fluo-4 to allow measurement of the luminal-free [Ca²⁺] (12). Cells were then permeabilized by incubation with saponin (10 μg/ml) and immobilized in 96-well plates to allow continuous monitoring of the Ca²⁺ content of the intracellular stores using a FlexStation fluorescence plate reader (MDS Analytical Devices) (12). All experiments were performed at 20 °C in cytosol-like medium with a free [Ca²⁺] of 220 nm (CLM: 140 mm KCl, 20 mm NaCl, 1 mm EGTA, 375 μm CaCl₂, 20 mm Pipes, pH 7.0). MgATP (1.5 mm) was added to allow Ca²⁺ uptake into the stores, and when steady-state loading had been achieved, IP₃ was added with thapsigargin (1 μm) to allow Ca²⁺ release to be measured without further re-uptake. IP₃-evoked Ca²⁺ release is expressed as a fraction of the Ca²⁺ released by addition of ionomycin (1 μm) (12). Similar methods were used to measure IP₃-evoked Ca²⁺ release from Sf9 cells expressing IP₃R2.

Binding of ³H-IP₃ to IP₃R2

Membranes were prepared from Sf9 cells expressing mouse IP₃R2 as described (17). Briefly, cells were harvested 60 h after infection, washed, and resuspended in Ca²⁺-free CLM supplemented with complete protease inhibitor mixture (Roche Applied Science, 1 tablet/25 ml). The suspension (3 × 10⁷ cells/ml) was homogenized and centrifuged (130,000 × g, 60 min), and the pellet was resuspended in Ca²⁺-containing CLM and stored at −80 °C. Membranes (72 μg of protein/ml) were resuspended in CLM (0.5 ml, supplemented with 2 mm MgCl₂) containing [³H]IP₃ (1.5 nm, 18 Ci/mmol) and appropriate concentrations of unlabeled IP₃. Variations in the composition of CLM at different steps were dictated by the need to minimize proteolysis during lysis (bivalent cation-free CLM) and then to include both Ca²⁺ and Mg²⁺ in the [³H]binding assay more effectively to mimic the composition of cytosol and for comparison with our previous analyses of [³H]IP₃ binding to IP₃R1 and IP₃R3 (16).

After 5 min at 2 °C, during which equilibrium was attained, the incubations were stopped by addition of γ-globulin (30 μl, 25 mg/ml) and polyethylene glycol 8000 (500 μl, 30%). After centrifugation (20,000 × g for 5 min at 2 °C), pellets were dissolved in Triton X-100 (200 μl, 2%), and ³H activity was determined by liquid scintillation counting after addition of Ecoscint A scintillation mixture (1 ml, National Diagnostics). Total [³H]IP₃ binding was typically 2300 dpm, of which ∼90% was specific binding. Results were fitted to Hill equations (GraphPad Prism, version 5) from which the half-maximal inhibitory concentration, and thereby the K_D, were determined.

Measurements of cAMP and [Ca²⁺]_i in Intact Cells

HEK-PR1 cells in 96-well plates were cultured for 2–3 days until almost confluent, washed, loaded with fluo-4, and [Ca²⁺]_i was then measured using a FlexStation fluorescence plate reader as described before (7). All experiments were performed at 20 °C in HBS or Ca²⁺-free HBS. HBS had the following composition: 135 mm NaCl, 5.9 mm KCl, 1.2 mm MgCl₂, 1.5 mm CaCl₂, 11.6 mm HEPES, 11.5 mm glucose, pH 7.3; Ca²⁺-free HBS was supplemented with 10 mm BAPTA.

Single cell analyses of [Ca²⁺]_i in fura-2-loaded HEK-PR1 cells were performed as previously reported, with fluorescence ratios calibrated, after correction for background fluorescence, to [Ca²⁺]_i by reference to Ca²⁺-calibration solutions (18).

For assays of cAMP, HEK-PR1 cells in 24-well plates were cultured for 2–3 days (as above) until near confluence and then stimulated under identical conditions to those used for measurements of [Ca²⁺]_i; the only difference was the omission of fluo-4-AM and Pluronic F-127 from the 1-h loading incubation. Cell extracts were prepared, and their cAMP content was determined by radioimmunoassay using acetylated standards prepared from a cAMP stock calibrated by its UV absorption (ϵ₂₅₈ = 14,100) as described (7, 19). It is important to note that single cell measurements of [Ca²⁺]_i established that 98% of cells responded to CCh, and 99% of those responded to PTH (7). This observation justifies our use of cell populations for comparisons of the effects of stimuli on [Ca²⁺]_i and cAMP levels.

Nuclear Patch Clamp Recording

Currents were recorded from patches excised from the outer nuclear envelope of DT40-KO cells stably expressing mouse IP₃R2, using Cs⁺ as the charge carrier. Bath and pipette (PS) solutions had the following composition: 200 mm CsCH₃SO₃, 500 μm BAPTA, 211 μm CaCl₂ (free [Ca²⁺] = 200 nm), 10 mm HEPES, pH 7.2 (with CsOH). Where appropriate, IP₃, cAMP, and/or ATP were included in PS. Data were collected and analyzed exactly as described previously (20).

Analysis

Concentration-effect relationships for each experiment were individually fitted to Hill equations using non-linear curve-fitting (GraphPad Prism, version 5), and the results obtained from each (EC₅₀, Hill coefficient h, and maximal response) were pooled for analysis and presentation. For simplicity, EC₅₀ values are reported as means ± S.E., although log EC₅₀ values were used for statistical analysis. Student's t test or one-way analysis of variance followed by post hoc Bonferroni test were used as appropriate.

Materials

Sources of materials not specified herein are provided in a previous study (7).

RESULTS AND DISCUSSION

Potentiation of IP₃-evoked Ca²⁺ Release by cAMP

CCh, which activates phospholipase C and thus IP₃ formation via endogenous muscarinic acetylcholine receptors, stimulated Ca²⁺ release from the intracellular stores of HEK-PR1 cells (Fig. 1B). The response was potentiated by forskolin, which activates AC directly (7), or by PTH, which activates AC via heterologously expressed type 1 PTH receptors and the G-protein, G_s (Fig. 1B). The effect of PTH on CCh-evoked Ca²⁺ release was mimicked by a membrane-permeant analogue of cAMP, 8-Br-cAMP (EC₅₀ = 324 μm) (7), and the sensitivity to 8-Br-cAMP was unaffected by H89 at a concentration (10 μm) sufficient to inhibit fully PKA-mediated protein phosphorylation (Fig. 1C) (7). Neither forskolin nor 8-Br-cAMP alone evoked Ca²⁺ release (7).

In permeabilized HEK-PR1 cells, IP₃ stimulated release of Ca²⁺ from the intracellular stores, cAMP increased the sensitivity to IP₃, and the effects of cAMP were again unaffected by inhibition of PKA (Fig. 1D). These results confirm our earlier conclusion that in HEK-PR1 cells, PTH potentiates IP₃-evoked Ca²⁺ release via cAMP and with no requirement for PKA (7). The earlier work established that IP₃R2 and AC6 are selectively associated and both are required for PTH to potentiate CCh-evoked Ca²⁺ signals (7) (Fig. 1A). But it is not yet clear whether the specific requirement for IP₃R2 derives entirely from its association with AC6 or is IP₃R2 also the only subtype of IP₃R to respond directly to cAMP?

Regulation of All IP₃R Subtypes by cAMP

IP₃ stimulated release of Ca²⁺ from the intracellular stores of permeabilized DT40 cells stably expressing mammalian IP₃R1, IP₃R2, or IP₃R3 (Fig. 2A), but not from the parental cells lacking endogenous IP₃R (not shown) (21, 22). Because the three IP₃R subtypes are not expressed at identical levels (Fig. 2B and Table 1), we cannot assume that the different fractions of the intracellular Ca²⁺ stores released by a maximal concentration of IP₃ or the different EC₅₀ values (Table 1) directly reflect specific properties of the three IP₃R subtypes. It is generally suggested that IP₃R2 is more sensitive than IP₃R1 to IP₃, and both are considerably more sensitive than IP₃R3 (23, 24). However, in our assays of DT40 cells stably expressing a single IP₃R subtype we found the order of sensitivity to be: IP₃R1 > IP₃R2 ≫ IP₃R3 (Table 1). The slightly lower than anticipated sensitivity of cells expressing IP₃R2 probably results from IP₃R2 being expressed at a lower level (∼30–40%) than the other subtypes (Table 1) (21). The ∼5-fold difference in IP₃ sensitivity between cells expressing IP₃R1 and IP₃R3 does not detract from our ability to resolve the effects of cAMP on IP₃-evoked Ca²⁺ release via each IP₃R subtype.

FIGURE 2.

cAMP potentiates IP₃-evoked Ca²⁺ release by the three subtypes of IP₃R. A, IP₃-evoked Ca²⁺ release from the intracellular stores of DT40 cells expressing only IP₃R1, IP₃R2, or IP₃R3. B, immunoblots (with AbC) show levels of IP₃R expression in each of the three cell lines (95 μg of protein/lane) compared with membranes from rat cerebellum (1.1 and 0.2 μg/lane); the position of the 220-kDa M_r marker is shown. The blot is typical of six experiments from two different preparations. Summary results are shown in Table 1. C, typical results from permeabilized DT40 cells expressing the indicated IP₃R incubated with ATP (added at the first arrow) to allow loading of the intracellular Ca²⁺ stores, before addition (second arrow) of cAMP alone (i, 1 mm), IP₃ alone (ii, 30 nm for IP₃R1 and IP₃R2; 300 nm for IP₃R3), or IP₃ with cAMP (iii). Each trace is the average of three to four wells from a single experiment and is representative of results from three to four independent experiments. D, from experiments similar to those shown in C, the concentration-dependent effects of cAMP on the EC₅₀ for IP₃-evoked Ca²⁺ release are shown for each IP₃R subtype. Results are means ± S.E., n ≥ 3.

TABLE 1.

Responses of IP₃R subtypes to IP₃ and cAMP

Permeabilized DT40 cells expressing only a single mammalian IP₃R subtype were used to examine the sensitivity of the intracellular Ca²⁺ stores to IP₃ (EC₅₀) and the fraction of the stores released by a maximally effective concentration of IP₃. For each cell line, IP₃R expression was quantified using immunoblotting with AbC (Fig. 2B), which interacts equally with all three IP₃R subtypes (16) and calibrated against a stock of rat cerebellar membranes for which B_max (tetrameric IP₃R/10⁶ cells) had been determined from [³H]IP₃ binding. The effects of a maximally effective concentration of cAMP on the EC₅₀ for IP₃-evoked Ca²⁺ release are shown, derived from experiments similar to those shown in Figs. 1D and 2. The sensitivity to cAMP is shown by the EC₅₀ for the effects of cAMP on IP₃-evoked Ca²⁺ release. Results are means ± S.E. (n ≥ 3).

	IP3R1	IP3R2	IP3R3	IP3R2-ΔATPB
IP3R expression (fmol/106 cells)	38 ± 2	16 ± 1	56 ± 5	NDa
EC50 for IP3 (nm)	56 ± 3	74 ± 10	255 ± 9	246 ± 17
EC50 for IP3 after cAMP (nm)	13 ± 1	22 ± 1	134 ± 19	44 ± 3
Fold stimulation by cAMP	4.2 ± 0.1	3.3 ± 0.3	2.0 ± 0.3	5.7 ± 0.6
Maximum response to IP3 (%)	83 ± 10	80 ± 1	80 ± 1	45 ± 4
Maximum response to IP3 after cAMP (%)	79 ± 2	81 ± 1	82 ± 1	53 ± 4
EC50 for cAMP (μm)	642 ± 158	267 ± 80	361 ± 74	ND

^a ND, not determined.

We used the three DT40 cell lines to examine the effects of cAMP on IP₃-evoked Ca²⁺ release. The results demonstrate that cAMP alone had no effect on the intracellular Ca²⁺ stores (Fig. 2C), but it significantly increased the sensitivity of each IP₃R subtype to IP₃ (Fig. 2, C and D). For all subtypes, a maximally effective concentration of cAMP caused the EC₅₀ for IP₃-evoked Ca²⁺ release to decrease by between 2- and 4-fold (Table 1). This increase in sensitivity is similar to the ∼3-fold increased sensitivity to CCh of HEK-PR1 cells treated with PTH (7), but less than the ∼7-fold increase in IP₃ sensitivity evoked by a maximal concentration of cAMP in permeabilized HEK cells (Fig. 1D). There was no statistical difference (p < 0.05) between either the sensitivities of the three IP₃R subtypes to cAMP (EC₅₀) or the Hill coefficients of the concentration-effect relationships. We conclude that the three subtypes of IP₃R are similarly sensitive to cAMP, with EC₅₀ values between ∼300 and 650 μm (Table 1).

Interactions of cAMP with IP₃R2

Our earlier work established the importance of IP₃R2 in mediating responses to PTH (7) and responses of homomeric IP₃R2 are potentiated by cAMP (Fig. 2, C and D), we therefore focused on this IP₃R subtype for our further analyses of the interactions of cAMP with IP₃R.

It proved impracticable to measure [³H]IP₃ binding in CLM to membranes prepared from DT40-IP₃R2 cells; we therefore used membranes from Sf9 cells expressing the same mouse IP₃R2 (Fig. 3A). We first confirmed that IP₃R2 expressed in Sf9 cells are regulated by cAMP. Whereas IP₃ (10 μm) stimulated release of only 7 ± 1% (EC₅₀ = 500 ± 50 nm) of the Ca²⁺ stores of uninfected Sf9 cells, it released 30 ± 4% (EC₅₀ = 122 ± 35 nm) of the stores from permeabilized Sf9 cells expressing mouse IP₃R2 (Fig. 3B). This confirms that most IP₃-evoked Ca²⁺ release from the infected Sf9 cells is mediated by heterologously expressed IP₃R2. Treatment with cAMP (1 mm), which itself caused no Ca²⁺ release, increased the sensitivity of the expressed IP₃R2 to IP₃: the EC₅₀ was reduced from 122 ± 35 nm to 41 ± 12 nm (Fig. 3B). These results establish that IP₃R2 expressed in Sf9 cells, like those expressed in HEK (7) or DT40 cells (Fig. 2), are sensitized to IP₃ by cAMP.

FIGURE 3.

Interaction of cAMP with IP₃R2 expressed in Sf9 cells. A, immunoblot (with AbC) showing expression of IP₃R2 in membranes from infected Sf9 cells, but not from membranes of uninfected cells (control). Protein loadings are shown in micrograms. B, concentration-dependent release by IP₃ of Ca²⁺ from permeabilized Sf9 cells expressing mouse IP₃R2 with or without addition of cAMP (1 mm). C, specific binding of [³H]IP₃ (1.5 nm) to membranes from Sf9 cells expressing mouse IP₃R2 in the presence of the indicated concentrations of unlabeled IP₃. There was no detectable specific binding of [³H]IP₃ to membranes from uninfected cells. Because the concentration of [³H]IP₃ (1.5 nm) was less than the K_D for IP₃ (4.3 nm), a change in K_D or B_max would be reflected in the specific [³H]IP₃ binding. The open circle shows that cAMP (10 mm) had no effect on specific [³H]IP₃ binding. Results (B and C) show means ± S.E., n ≥ 3.

IP₃ bound to IP₃R2 expressed in Sf9 cells with high affinity (K_D = 4.30 ± 0.04 nm, B_max = 2.55 pmol/mg), but specific binding of [³H]IP₃ (1.5 nm) was unaffected by cAMP (10 mm) (Fig. 3C). This demonstrates that the effect of cAMP on IP₃R2 is not mediated by an increase in its affinity for IP₃, but must instead result from an increase in the effectiveness with which IP₃ binding is coupled to channel opening; cAMP increases the efficacy of IP₃.

Effects of cAMP on Single IP₃R2

In patch clamp recordings from excised nuclear patches of DT40-IP₃R2 cells, IP₃ (100 μm) in the absence of ATP (which is not required for high concentrations of IP₃ to maximally activate IP₃R2 (25)) stimulated opening of large-conductance cation channels (γ_Cs ∼200 pS, Fig. 4, A, i, and ii, and B). These channels were absent from the nuclei of DT40-KO cells (Fig. 4A, iii). The single channel open probability (P_o) of these IP₃R was similar (P_o = 0.49 ± 0.07) (Fig. 4A, ii) to that reported for maximally stimulated IP₃R2 expressed in the plasma membrane (25), or nuclear IP₃R1 and IP₃R3 (20). The latter recordings used K⁺ as charge-carrier, but P_o for IP₃R1 is similar whether K⁺ (P_o = 0.45 ± 0.07) or Cs⁺ (P_o = 0.41 ± 0.04) is used as the charge carrier. These results establish that 100 μm IP₃ in the absence of ATP maximally activates IP₃R2.

FIGURE 4.

Effects of cAMP on single channel activities of IP₃R2. A, traces show excised nuclear patch clamp recordings from DT40-IP₃R2 (i and ii) or DT40-KO cells (iii) in which IP₃ (100 μm) was included in PS as indicated. B, current-voltage (i-V) relationship for single IP₃R2 stimulated with IP₃ (1 μm), with or without cAMP (1 mm) in PS. The single channel slope conductances (γ_Cs) were 204 ± 6 and 200 ± 12 pS in the absence and presence of cAMP, respectively. C, typical traces from single IP₃R2 stimulated with submaximal IP₃ (1 μm) in the presence and absence of cAMP (1 mm) and/or ATP (5 mm) in PS as indicated. D, from recordings similar to those shown in D, NP_o is shown for IP₃R2 stimulated as indicated. Note the use of NP_o to report channel activity here because the IP₃Rs are less active in some of the recording conditions, making it difficult to determine reliably the number of IP₃R in each patch (see text). For all traces, PS contained a free [Ca²⁺] of 200 nm, the holding potential was +40 mV, C denotes the closed state, and each trace is typical of at least three similar recordings. Results (B and D) are means ± S.E., n ≥ 3.

The effects of cAMP are most evident in Ca²⁺ release assays after submaximal activation with IP₃ (Fig. 1D and Table 1) (7). Our initial examination of the effects of cAMP on single IP₃R2 were therefore performed without ATP and with a submaximal concentration of IP₃. Because P_o is much lower under these conditions, it is impossible to resolve with confidence the number of active IP₃R within a patch (20) and so impossible to calculate P_o. We therefore use NP_o (the overall channel activity) to report the activity of IP₃R in these submaximally stimulated patches. A concentration of cAMP (1 mm) sufficient to sensitize maximally IP₃-evoked Ca²⁺ release (Fig. 2D) had no effect alone on channel activity recorded from nuclei of DT40-IP₃R2 cells (Fig. 4C, i). But cAMP significantly increased the activity of IP₃R2 stimulated with a submaximal concentration of IP₃ (1 μm): NP_o increased from 0.06 ± 0.02 (n = 11) in the absence of cAMP to 0.18 ± 0.05 (n = 5) in its presence (Fig. 4, C, ii and iii, and D). The single channel Cs⁺ conductance was unaffected by cAMP (Fig. 4B). Because these experiments were performed in the absence of ATP, the effects of cAMP cannot be attributed to PKA-mediated phosphorylation of IP₃R.

In keeping with work from others (25), ATP (5 mm) alone had no effect on IP₃R2 activity (not shown), but it potentiated responses to a submaximal concentration of IP₃ (1 μm): NP_o increased from 0.06 ± 0.02 (n = 11) to 0.15 ± 0.02 (n = 5) in the presence of ATP (Fig. 4, C, ii, and iv, and D). In the presence of this maximally effective concentration of ATP (25), cAMP (1 mm) further potentiated responses to IP₃ (1 μm): NP_o increased from 0.15 ± 0.02 to 0.41 ± 0.05 (n = 6) in the presence of cAMP (Fig. 4, C, iv and v, and D). In keeping with our results from Ca²⁺ release assays (Table 1), cAMP did not increase NP_o of IP₃R2 stimulated with a maximally effective concentration of IP₃ (100 μm): NP_o was 0.49 ± 0.07 and 0.51 ± 0.1 (n = 3) in the absence and presence of cAMP, respectively (Fig. 4D). We conclude that cAMP, either directly or via a tightly associated accessory protein, increases the activity of IP₃R2 stimulated with IP₃.

cAMP Potentiation of IP₃-evoked Ca²⁺ Release Is Not Mediated by the Modulatory ATP-binding Site

We further considered the possibility that the effects of cAMP might reflect its interaction with the site(s) through which ATP potentiates IP₃R function (25, 26). This seems unlikely from the results of our single channel analyses (Fig. 4) and because cAMP potentiates responses to IP₃ in the presence of 1.5 mm ATP (Fig. 2, C and D), which others have shown to be more than sufficient to potentiate maximally responses to IP₃ (25). Yule and co-workers have established that an ATP-binding site in IP₃R2 (ATPB, residues 1969–1974) mediates modulation of IP₃R2 gating by ATP. Mutation of glycine residues (G1969A, G1971A, and G1974A) within this site abolishes the potentiating effect of ATP on IP₃-evoked Ca²⁺ release (25). We used the same cell line (DT40-ΔATPB), kindly provided by David Yule (University of Rochester), to examine the effects of cAMP on IP₃-evoked Ca²⁺ release. Responses to IP₃ in these DT40-ΔATPB cells were also potentiated by cAMP (Fig. 5, A and B, and Table 1). These results, together with those from single channel analyses (Fig. 4), establish that the effects of cAMP on IP₃R2 are not mediated by its interaction with the site through which ATP potentiates activity.

FIGURE 5.

Potentiation of IP₃R2 by cAMP does not require the ATPB-binding site. A, typical results from permeabilized DT40 cells expressing only IP₃R2-ΔATPB incubated with ATP (added at the first arrow) before addition (second arrow) of cAMP alone (i, 3 mm), IP₃ alone (ii, 300 nm), or IP₃ with cAMP (iii). Each trace is the average of three to four wells from one experiment and is representative of results from three independent experiments. B, summary showing the concentration-dependent effects of IP₃ on Ca²⁺ release in the absence and presence of cAMP (3 mm). Results are means ± S.E., n ≥ 3.

PKA-dependent and -independent Regulation of IP₃R by cAMP

All three IP₃R subtypes respond directly to cAMP (Fig. 2), all three can also be phosphorylated, at multiple sites, by PKA (6, 7, 27–34), and substantial evidence suggests that the phosphorylation can modulate IP₃R activity (28, 31, 35–38). Phosphorylation of IP₃R1 by PKA increases their activity (27, 31, 32, 39–41). An initial suggestion that PKA inhibits IP₃R1 probably resulted from a counteracting stimulatory effect of PKA on Ca²⁺ re-uptake (30). IP₃R2 appears to be a rather poor substrate for PKA (7, 27, 28, 36), although it is phosphorylated at Ser-937 (34), and in cells expressing predominantly IP₃R2, PKA typically causes a very modest increase in their sensitivity to IP₃ (7, 28, 35, 42). In DT40 cells expressing only IP₃R2 forskolin enhances responses to IP₃, but the extent to which this requires activation of PKA is unclear. When a PKA-selective analog of cAMP was used, phosphorylation of Ser-937 by PKA was required for potentiation of the Ca²⁺ signals evoked by threshold concentrations of CCh (34). Clearly, therefore, PKA can both phosphorylate and modestly sensitize IP₃R2; the extent to which that underlies the effects of forskolin or cAMP is unresolved. The effects of PKA on IP₃R3 are less clear. Analyses from cells in which IP₃R3 is the major subtype suggest that PKA either modestly sensitizes them to IP₃ (28) or attenuates their responses (33, 43). The only published analysis of homomeric IP₃R3 expressed in DT40 cells concluded that PKA had no effect on IP₃-evoked Ca²⁺ release (27).

Under conditions where the catalytic subunit of PKA (200 units/ml for 10 min) phosphorylates IP₃R in HEK-PR1 cells (7), it had no effect on IP₃-evoked Ca²⁺ release from DT40 cells expressing IP₃R2 (Fig. 6A) or IP₃R3 (not shown), and it only very modestly increased the sensitivity of IP₃R1 to IP₃ (the EC₅₀ fell from 36 to 27 nm (supplemental Fig. S1, A and B). We note that, even for IP₃R1, where the effects of PKA are relatively uncontentious, phosphorylation by PKA typically increases the IP₃ sensitivity by <2-fold (28, 36, 40). In our analyses of HEK-PR1 cells also, the very modest stimulatory effect of PKA on IP₃-evoked Ca²⁺ release (∼30%) is much smaller than that of cAMP (∼450%) (7).

FIGURE 6.

Sensitization of IP₃R2 by cAMP does not require activation of PKA. A, IP₃-evoked Ca²⁺ release from the intracellular stores of permeabilized DT40-IP₃R2 cells with and without preincubation with the catalytic subunit of PKA (200 units/ml, 10 min). B, Ca²⁺ release from permeabilized DT40-IP₃R2 cells stimulated with IP₃ alone (60 nm) or in combination with cAMP (3 mm) after the indicated pretreatments (10 min) with the catalytic subunit of PKA (200 units/ml) and/or H89 (10 μm). Results are means ± S.E., n ≥ 3.

In an attempt to reduce phosphorylation by endogenous PKA (41), we first incubated DT40-IP₃R2 cells with H89 (10 μm, 60 min) to inhibit PKA before permeabilizing the cells and assessing their responses to IP₃. Here too, there was no detectable effect of the catalytic subunit of PKA on IP₃-evoked Ca²⁺ release via IP₃R2 (supplemental Fig. S1C). Under the same conditions, where direct activation of PKA had no effect on IP₃-evoked Ca²⁺ release, cAMP (3 mm) potentiated the response to a submaximal concentration of IP₃ (60 nm) to the same extent whether applied alone, or after pretreatment (10 min) with H89 (10 μm), the catalytic subunit of PKA (200 units/ml) or both (Fig. 6B). We conclude that sensitization of IP₃R2 by cAMP occurs independently of activation of PKA.

We can only speculate on the negligible (IP₃R1) or undetectable effect (IP₃R2 and IP₃R3) of PKA on IP₃-evoked Ca²⁺ release from DT40 cells expressing homomeric IP₃R. Association of IP₃R with AKAP facilitates their phosphorylation by endogenous PKA (40, 44, 45), but ineffective targeting of PKA by AKAP is unlikely to prevent exogenous catalytic subunit of PKA from phosphorylating IP₃R (Fig. 6 and supplemental Fig. S1). Accessory proteins, perhaps analogous to the IP₃R-associated cGMP kinase substrate (IRAG) required for regulation of IP₃R by cGMP-dependent protein kinase (46), may be required for PKA to modulate IP₃R activity. IP₃R may already be phosphorylated in DT40 cells, although the lack of effect of PKA in permeabilized cells after sustained pretreatment of intact cells with an inhibitor of PKA suggests this is unlikely (supplemental Fig. S1C). Furthermore, even when IP₃R3 was shown to be phosphorylated by PKA in DT40 cells, there was no change in their response to IP₃ (27). A further possibility is that an active protein phosphatase is associated with IP₃R (32, 47) and limits their steady-state phosphorylation in DT40 cells.

The essential point for the present discussion is that cAMP directly potentiates IP₃-evoked Ca²⁺ release and the effect is both more substantial than, and independent of, the effects of phosphorylation by PKA.

Loss of Gα_s Attenuates Sensitization of IP₃ Receptors by PTH

Potentiation of CCh-evoked Ca²⁺ signals by forskolin or PTH requires an intimate association between IP₃R2 and AC6 that allows cAMP to pass directly to the IP₃R (7) (Fig. 1A). Subsequent experiments seek to establish whether Gα_s, which links PTH receptors to activation of AC, is also specifically associated with these AC·IP₃R junctions.

We established seven stable HEK-PR1 cell lines in which shRNA was used to reduce expression of Gα_s (HEK-PR1α_s⁻ cells) and three mock transfected lines. The responses of the latter to CCh alone or with PTH were indistinguishable from the parental cells (not shown). Results are shown for only one of the HEK-PR1α_s⁻ lines, in which Gα_s expression was reduced by >95% (7). The concentration-dependent effects of CCh alone or with PTH on Ca²⁺ signals were similar in each of the other HEK-PR1α_s⁻ lines. Neither the Ca²⁺ signals evoked by CCh alone (7), their potentiation by forskolin (Fig. 7A), nor forskolin-stimulated cAMP production (Fig. 7B) was affected by loss of Gα_s. This establishes that selection of the stable HEK-PR1α_s⁻ cells had no effect on AC activity, CCh-mediated Ca²⁺ signaling, or potentiation of IP₃R activity by cAMP.

FIGURE 7.

Loss of Gα_s attenuates PTH-potentiated Ca²⁺ signals. A and B, concentration-dependent effects of forskolin on the potentiation of the Ca²⁺ release evoked by CCh (1 mm) (A) and on the increase in cAMP production measured 30 s after stimulation of HEK-PR1 and HEK-PR1α_s⁻ cells (B). C, concentration-dependent effects of PTH on cAMP formation (measured after 30 s) in HEK-PR1 and HEK-PR1α_s⁻ cells alone or after pre-incubation (20 min) with SQ 22536 (SQ, 1 mm) and 2′,5′-dideoxyadenosine (DDA, 200 μm) (SQ/DDA). D, cells treated as in C (with the same symbols) but showing the effects of PTH on CCh-evoked Ca²⁺ signals. Results (A–D) are means ± S.E., n ≥ 3.

To allow direct comparison between PTH-evoked Ca²⁺ and cAMP signals, we measured both responses under the same conditions, after identical intervals (30 s) and in the absence of 3-isobutylmethylxanthine (see “Experimental Procedures”). Under these conditions, PTH caused a concentration-dependent stimulation of AC activity in both HEK-PR1 and HEK-PR1α_s⁻ cells (Fig. 7C). Loss of Gα_s caused the EC₅₀ for PTH-evoked cAMP formation to increase by at least 10-fold (Fig. 7C), while the maximal response (to 1 μm PTH) was reduced by 61 ± 7% and then reduced further (by 90 ± 5%) when AC was inhibited by SQ 22536 (1 mm) with DDA (200 μm) (SQ/DDA). In HEK-PR1 cells, inhibition of AC by SQ/DDA more fully inhibited PTH-evoked cAMP formation (81 ± 5%) than did loss of Gα_s (61 ± 7%) (Fig. 7C).

Inhibition of AC alone had no effect on PTH-potentiated Ca²⁺ signals (Fig. 7D) (7), but loss of Gα_s reduced their peak amplitude without affecting their sensitivity to PTH (Fig. 7D and Table 2). We confirmed, using single cell analyses of [Ca²⁺]_i, that the same fraction of cells responded to PTH in normal and Gα_s-deficient cells, that the two cell lines responded similarly to CCh alone, and that the amplitude of the PTH-potentiated CCh-evoked Ca²⁺ signal was decreased by 29 ± 4% in HEK-PR1α_s⁻ cells (Table 2). This demonstrates that the results with cell populations, where loss of Gα_s diminished peak PTH-evoked Ca²⁺ signals by 34 ± 3% (Fig. 7D), do not arise from an all-or-nothing loss of responsiveness of individual cells. For each cell, therefore, loss of >95% of Gα_s caused a ∼30% decrease in the peak Ca²⁺ signals evoked by PTH.

TABLE 2.

Potentiation of CCh-evoked Ca²⁺ signals by PTH in Gα_s-deficient cells

The EC₅₀ values for potentiation of CCh-evoked Ca²⁺ release by PTH or forskolin in populations of HEK-PR1 cells are shown. Results are means ± S.E., n ≥ 3. Single cells loaded with fura-2 were stimulated with CCh alone (1 mm) followed by PTH (100 nm) in the continued presence of CCh. Responses from 6–12 independent coverslips, each including 116–233 cells, were analyzed. The results show the increase in [Ca²⁺]_i and the fraction of responsive cells in HEK-PR1 and HEK-PR1α_s⁻ cells.

	HEK-PR1	HEK-PR1αs−
Cell populations
PTH	52 ± 10 nm	62 ± 11 nma
PTH + H89	NDb	46 ± 12 nm
PTH + SQ/DDA	NDb	219 ± 27 nma
Forskolin	30 ± 6 μm	28 ± 5 μm
Single cells
Initial response to CCh, Δ[Ca2+]i	585 ± 17 nm	593 ± 13 nm
PTH then CCh, Δ[Ca2+]i	257 ± 32 nmc	183 ± 11 nmc
Cells responding to PTH then CCh	95 ± 1%	91 ± 3%

^a p < 0.05 (between marked values).

^b ND, not determined.

^c p < 0.05 (between marked values).

These results establish that the ability of all concentrations of PTH to potentiate CCh-evoked Ca²⁺ signals is insensitive to a uniform ∼80% inhibition of AC activity by low affinity inhibitors of AC (SQ/DDA). But a lesser inhibition of AC arising from loss of Gα_s attenuates PTH-mediated Ca²⁺ signaling (Fig. 7D). Why should inhibition of AC activity by loss of Gα_s more effectively inhibit PTH-mediated Ca²⁺ signaling than direct inhibition of AC by low affinity inhibitors?

Association of Gα_s with AC·IP₃R Junctions

Earlier experiments comparing the effects of inhibiting AC by reducing its expression with RNA interference or by inhibition of AC with SQ/DDA revealed a similar phenomenon to that observed with loss of Gα_s. Complete inhibition of AC within individual AC·IP₃R junctions by selective inhibition of AC6 expression attenuated PTH-evoked Ca²⁺ signaling. However, a greater, but uniformly distributed, inhibition by low affinity inhibitors of AC (SQ/DDA) had no effect on Ca²⁺ signaling (7). These and related results lead to our conclusion that the AC·IP₃R junction (Fig. 1A) is the minimal signaling unit and that within it cAMP is delivered to IP₃R at concentrations greater than are needed maximally to sensitize associated IP₃R. Each junction operates with a substantial “safety margin.” Removing AC from individual junctions (by inhibition of AC expression) incapacitates individual junctions, whereas SQ/DDA uniformly attenuates cAMP formation at each junction, but the lesser amount of cAMP made within each is still sufficient to sustain effective communication between AC and IP₃R (7).

Attenuation of the responses to PTH by loss of Gα_s might result entirely from the diminished activity of AC, or it might reflect an additional stimulatory role for Gs in modulating responses of IP₃R to cAMP. We previously demonstrated that the maximal effects of 8-Br-cAMP and PTH on the sensitivity of Ca²⁺ release to CCh were the same, and the two stimuli together had no greater effect (7). This provided persuasive evidence that cAMP alone mediates the effects of PTH on IP₃R sensitivity. That conclusion is further supported by the results shown in Fig. 8A, in which we compare the amounts of cAMP associated with comparable Ca²⁺ signals evoked by PTH under different conditions. The comparison is reasonable, because cAMP and [Ca²⁺]_i are measured at the same time (30 s) under identical conditions, and single cell analyses confirm that all cells respond similarly (Table 2). Comparably potentiated Ca²⁺ signals are associated with larger amounts of cAMP in normal HEK-PR1 cells than in HEK-PR1α_s⁻ cells. These results, where cells with most Gα_s appear least sensitive to cAMP, are inconsistent with Gα_s potentiating the responses of IP₃R to cAMP. These results reinforce our initial conclusion that cAMP alone mediates the effects of PTH on IP₃R (7).

FIGURE 8.

Association of Gα_s with AC·IP₃R junctions. A, from the results shown in Fig. 7, C and D, the relationships between the change in cAMP content and potentiation of CCh-evoked Ca²⁺ signals by PTH are shown for HEK-PR1 and HEK-PR1α_s⁻ cells alone or after pretreatment with SQ/DDA (20 min). The right panel shows an enlarged version of the relationship for the lower ΔcAMP levels. Results are means ± S.E., n ≥ 3. B, proposed arrangement of AC·IP₃R junctions. The central panel shows the signaling proteins that regulate the AC·IP₃R junction, allowing AC6 to deliver super-saturating concentrations of cAMP to an associated IP₃R2. The predicted consequences of incompletely inhibiting AC with low affinity inhibitors (SQ/DDA, right) or diminishing AC activity by removal of Gα_s from AC·IP₃R junctions (shRNA, left) are shown. As long as some Gα_s remains associated with the junction, the cAMP safety margin allows effective signaling to the IP₃R (upper left), but complete removal of Gα_s from a junction incapacitates signaling (lower left). We speculate that ∼22 Gα_s associate with each AC·IP₃R junction (see text). Whether PTH receptors associate specifically with individual junctions has not yet been addressed.

Although cAMP is the only signal through which PTH communicates with IP₃R, the effective cAMP cannot be uniformly distributed, because otherwise the effects of inhibiting cAMP formation should be identical whatever step in the signaling pathway is targeted for inhibition. We speculate that a small number of Gα_s are specifically associated with each AC·IP₃R junction (Fig. 8B). Random removal of Gα_s from the signaling complex (by inhibition of Gα_s expression) inactivates signaling from PTH to a complex without Gα_s, whereas complexes with residual Gα_s continue to function, albeit with a lesser cAMP “safety margin” (Fig. 8B, upper left) than normal junctions. Hence, for HEK-PR1α_s⁻ cells, PTH-potentiated Ca²⁺ signals are associated with lesser overall increases in cAMP than in HEK-PR1 cells (Fig. 8A). The maximal potentiated Ca²⁺ signal evoked by PTH is decreased by 30% in HEK-PR1α_s⁻ cells, even though lesser amounts of cAMP in normal cells with AC inhibited by SQ/DDA are associated with undiminished Ca²⁺ signals (Fig. 7D). These results suggest that the ≥95% knockdown of Gα_s inactivates signaling by PTH to ∼30% of the AC·IP₃R junctions. Assuming that a single Gα_s is sufficient to mediate effective transmission to an AC·IP₃R junction and that the 95% loss of Gα_s is randomly distributed among the junctions, we predict (supplemental text) that there is an average of 1.1 Gα_s within each functional synapse of Gα_s-deficient cells, and ∼22 Gα_s/synapse in normal cells.

Conclusions: Structure and Function of AC·IP₃R Junctions

We have shown that all three IP₃R subtypes are directly regulated by cAMP binding either directly to the IP₃R or to a protein tightly associated with it (Figs. 1–4). The effect of cAMP, which increased the efficacy of IP₃ (Fig. 4), required neither PKA (Fig. 6) nor the site through which ATP modulated IP₃R2 activity (Figs. 4 and 5). In HEK cells, we have established that a specific association of IP₃R2 with AC6 allows cAMP to pass directly from AC to the IP₃R within an AC·IP₃R junction (Fig. 8B) (7). This “binary mode” of cAMP signaling (Fig. 1A) allows robust communication between cell surface receptors and IP₃R, because each AC·IP₃R junction operates as an on-off switch with a large safety margin. The similar sensitivity of all IP₃R to cAMP (Fig. 2) suggests that the specific requirement for IP₃R2 in HEK cells derives from IP₃R2 selectively associating with AC6 to form the junctions required to deliver cAMP at sufficient concentration to regulate IP₃R. Results from cells with much reduced expression of Gα_s (Figs. 7 and 8) suggest that small numbers of Gα_s (∼22 α_s/AC·IP₃R junction) may also associate with the AC·IP₃R junction (Fig. 8B).

IP₃ and cAMP are ubiquitous intracellular messengers, and interactions between them are common. Here we demonstrate another level of interaction in which cAMP itself sensitizes each IP₃R subtype by increasing the efficacy of IP₃. The interaction is not mediated by PKA, nor does it involve the ATP-binding site of the IP₃R. It remains to be resolved whether cAMP binds directly to IP₃R or to a tightly associated accessory protein. Within intact HEK cells, a selective association of IP₃R2 with AC6 to form an AC·IP₃R junction (Fig. 1A) allows cAMP preferentially to regulate IP₃R2. The G-protein, G_s, that couples PTH to stimulation of AC, appears also to selectively associate with the AC·IP₃R signaling junction. Aside from defining a novel interaction between cAMP and Ca²⁺ signaling pathways, our results highlight the importance of the spatial organization of both pathways in mediating effective signal transduction.