Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs

Abstract

The ability of inositol 1,4,5-trisphosphate receptors (IP3R) to precisely initiate and generate a diverse variety of intracellular Ca(2+) signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP3, Ca(2+), and ATP). However, the contribution of IP3R heterotetramerization to Ca(2+) signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation. Additionally, we examine the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca(2+) signals. Under conditions where key regulators of IP3R function are optimal for Ca(2+) release, we demonstrate that individual monomers within heteromeric IP3Rs contributed equally toward generating a distinct 'blended' sensitivity to IP3 that is likely dictated by the unique IP3 binding affinity of the heteromers. However, under suboptimal conditions where [ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP3R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP3R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP3-induced Ca(2+) signaling in cells expressing multiple IP3R isoforms.

Keywords: ATP binding; Concatemer; calcium; calcium channel; calcium intracellular release; inositol 1,4,5-trisphosphate (IP3); inositol trisphosphate receptor (InsP3R).

FIGURE 1.

IP₃R subtypes oligomerize to form heterotetramers in native tissues. Cleared lysates from bovine salivary gland were prepared using CHAPS lysis buffer. A, lysates were resolved on 5% SDS-PAGE and immunoblotted with α-R1, α-R2CT, or αR3. Lysates from DT40 3KO cells stably expressing R1, R2, or R3 were used as controls. B, bovine salivary gland lysates cleared through ultracentrifugation were subject to size exclusion chromatography. Alternate fractions were resolved on 5% SDS-PAGE and probed using α-R1 or α-R2CT. IP₃R immunoreactivity was quantified using ImageJ and normalized to peak immunoreactivity. C, fractions 9–11 corresponding to native IP₃R (∼1000–1200 kDa) were pooled at subject to co-immunoprecipitation using α-R1 and protein A/G beads. Immunoprecipitates (IP) were probed using α-R1 or α-R2CT. Lysates from DT40-3KO cells stably expressing R1, R2, or R3 were used as controls. Representative immunoblots are shown.

FIGURE 2.

IP₃R concatenated dimers are stably expressed in DT40 3KO cells, form tetramers, and are functionally indistinguishable from IP₃R monomers. A, lysates from DT40-3KO or DT40-3KO cells stably expressing IP₃R constructs were resolved on 4% SDS-PAGE and probed with α-R2NT or α-R3. B, lysates from DT40-3KO cells stably expressing IP₃R constructs were resolved on 3–12% native-PAGE^TM Novex gels. Immunoblots were processed using α-R2NT or α-R3. C, [³H]IP₃ binding curves of R1, R2, and R3 monomers, compared with R1R1, R2R2, and R3R3 homodimers, generated using homologous competitive binding assays. D, [IP₃]-response relationships of R1, R2, and R3 monomers, compared with R1R1, R2R,2 and R3R3 homodimers. High throughput permeabilized cell IICR assays were carried out using FlexStation 3. IP₃-mediated Ca²⁺ release was induced through addition of varying [IP₃] in the presence of 5 mm ATP and 200 nm free Ca²⁺. All values were normalized to the maximal release rate. Each point is the mean ± S.E. of eight wells from at least three experiments.

FIGURE 3.

IP₃R heterodimers exhibit blended IP₃ sensitivities at optimal [ATP] and [Ca²⁺]. [IP₃]-response relationships of R2R2 and R3R2 were compared with R2R2 and R3R3 (A) and R1R2 was compared with R1R1 and R2R2 (B). High throughput permeabilized cell IICR assays were carried out using FlexStation 3. IP₃-mediated Ca²⁺ release was induced through addition of varying [IP₃] in the presence of 5 mm ATP and 200 nm free Ca²⁺. All values were normalized to the maximal release rate. Each point is the mean ± S.E. of eight wells from at least three experiments.

FIGURE 4.

IP₃ binding of R3R2 heterodimer. [³H]IP₃ binding curves of R2R2, R3R3, and R3R2 homo- and heterodimers were generated using homologous competitive binding assays. All values were normalized to total specific binding. Each point is mean ± S.E. from three experiments.

FIGURE 5.

ATP regulatory properties of R2R2, R3R3, and R3R2 dimers. Ca²⁺ release through R2R2 (A–C), R3R3 (D–F), and R3R2 (G–I) was measured using single cell permeabilized IICR assays. A, [IP₃]-response curve shows 5 mm ATP potentiates IICR only at submaximal [IP₃], not at maximal [IP₃], in R2R2. D, [IP₃]-response curve shows ATP is required to maximally potentiate IICR at all [IP₃] for R3R3. G, [IP₃]-response curve for R3R2 shows 5 mm ATP potentiates IICR only at submaximal [IP₃], not at maximal [IP₃], comparable with R2R2. Each point is mean ± S.E. from at least three experiments. Traces show Ca²⁺ release events at 3 and 30 μm IP₃ for R2R2 (B and C), R3R3 (E and F), and R3R2 (H and I) in the presence (solid) or absence (dotted) of 5 mm ATP. Each trace is the average of ∼40–60 cells and was normalized to the average Δ340/380 of the 10 s prior to IP₃ application. *, p ≤ 0.05, Student's unpaired t test.

FIGURE 6.

ATP sensitivities of R2R2, R3R3, and R3R2 dimers. Ca²⁺ release through R2R2 (A), R3R3 (B), and R3R2 (C) was measured using single cell permeabilized IICR assays. Ca²⁺ release was stimulated using 1 μm IP₃ for R2R2, 10 μm IP₃ for R3R3, and 1 μm IP₃ for R3R2 at various [ATP]. Release rates were plotted to a [ATP]-response curve, and the sensitivity to ATP was determined using a unimodal logistic equation. Each point is mean ± S.E. from at least three experiments.

FIGURE 7.

R2 dictates the patterns of B cell receptor-induced Ca²⁺ signals in R3R2 heterodimers. Representative Fura-2 AM recordings of four individual DT40-3KO cells stably expressing R2R2 (A), R3R3 (B), R2R3 (D), and R3R2 (E) when stimulated with 1 μg/ml α-IgM. C, histogram of the percentage of responding cells producing a specific number of oscillatory transients, quantifiably showing R2R2 (red) and R3R3 (blue) to exhibit distinct oscillatory patterns. F, histogram of the percentage of responding cells producing a specific number of oscillatory transients show R2R3 (red) and R3R2 (blue) generate oscillatory patterns comparable with R2R2.

FIGURE 8.

Stoichiometry of ATP regulation in R2R2. A, lysates from DT40-3KO cells stably expressing R2, R2^Δ, R2R2, R2R2^Δ, and R2^ΔR2^Δ were resolved on 4% SDS-PAGE. Immunoblots were probed with α-R2NT. B and C, representative Fura-2 AM recordings of four individual DT40 3KO cells stably expressing R2^ΔR2^Δ (B) and R2R2^Δ (C), when stimulated with 1 μg/ml α-IgM. D, quantification of oscillatory patterns of R2R2 (red), R2^ΔR2^Δ (green), and R2R2^Δ (blue). E–J, Ca²⁺ release through R2^ΔR2^Δ (E–G) and R2R2^Δ (H–J) measured using single cell permeabilized IICR assays. E, [IP₃]-response curves show 5 mm ATP does not potentiate Ca²⁺ release in R2^ΔR2^Δ at any [IP₃]. H, 5 mm ATP potentiates Ca²⁺ release in R2R2^Δ at submaximal [IP₃]. Each point is mean ± S.E. from at least three experiments. Traces show Ca²⁺ release events at 1 and 10 μm IP₃ for R2^ΔR2^Δ (F and G) and R2R2^Δ (I and J) in the presence (red) or absence (black) of 5 mm ATP. Each trace is the average of ∼40–60 cells and was normalized to the average Δ340/380 of the 10 s prior to IP₃ application. *, p ≤ 0.05, Student's unpaired t test.

FIGURE 9.

Mutating ATPB in R3R2 and R1R2 heterodimers causes a loss of ATP regulatory ability. Ca²⁺ release through R3R2^Δ and R1R2^Δ was measured using single cell permeabilized IICR assay. A, [IP₃]-response curves show 5 mm ATP (black) does not potentiate IICR at any [IP₃]. Each point is mean ± S.E. from at least three experiments. B, traces show Ca²⁺ release events through R3R2^Δ at 1 μm (dotted) and 6 μm (solid) in the presence (black) or absence (gray) of 5 mm ATP. C, traces show Ca²⁺ release events through R1R2^Δ at 1 μm (dotted) and 10 μm (solid) in the presence (black) or absence (gray) of 5 mm ATP. D, histogram depicting Ca²⁺ release rate though R1R2^Δ at submaximal (1 μm) and maximal (10 μm) [IP₃] in the presence (black) or absence (gray) of 5 mm ATP. Each trace is the average of ∼40–60 cells and was normalized to the average Δ340/380 of the 10 s prior to IP₃ application.

FIGURE 10.

R1R1R1R2 concatenated heterotetramer exhibits R1-like properties. A, lysates from DT40-3KO cells stably expressing the described constructs resolved on 3% SDS-PAGE and probed with α-R1. B, representative Fura-2 AM recordings of four individual DT40 3KO cells stably expressing R1R1R1R2, when stimulated with 1 μg/ml α-IgM. C, quantification of oscillatory patterns of R1R1R1R2 (red), compared with R1R1 (green) and R2R2 (blue). D, histogram depicting Ca²⁺ release rate through R1R2 at submaximal (1 μm) and maximal (10 μm) [IP₃] in the presence (red) or absence (black) of 5 mm ATP. E, representative single channel recordings of R1R1R1R2 using on-nucleus configuration of the patch clamp technique, performed in the presence of 1 or 10 μm IP₃, 200 nm free Ca²⁺, and either 10 μm or 5 mm ATP. F, histogram depicting channel P_o for R1R1R1R2 at 10 μm or 5 mm ATP. G–J, Ca²⁺ release through R1R1 (G) and R1R1R1R1 (H–J) was measured using single cell permeabilized IICR assay. G, [IP₃]-response curves for R1R1 in the absence (black) or presence (red) of 5 mm ATP. H, histogram depicting Ca²⁺ release rate at submaximal (3 μm) and maximal (30 μm) [IP₃] in the presence (red) or absence (black) of 5 mm ATP. I and J, traces show Ca²⁺ release events at 3 μm (I) and 30 μm (J) IP₃ for R1R1R1R2. Each trace is the average of ∼40–60 cells and was normalized to the average Δ340/380 of the 10 s prior to IP₃ application.

FIGURE 11.

Novel insights into the function and regulation of IP₃R heterotetramers. Individual subtypes are represented as distinct colors as follows: R1, red; R2, blue; and R3, green. A, R3R2 heterodimers, which generate heterotetramers containing an equal proportion of R2 and R3, exhibit unique IP₃ binding properties (affinity, cooperativity) that are distinct to that of the two constituent subtypes. B, at optimal conditions for Ca²⁺ release (200 nm Ca²⁺, 5 mm ATP), heterodimers containing R2 and either R1 or R3 exhibit an IP₃ sensitivity intermediate to that of the two constituent subtypes that is potentially reflective of the heterodimers' unique IP₃ binding affinity. C, when ATP levels are reduced to submaximal concentrations, two R2 subunits are sufficient to dictate the regulatory properties of heterotetramers containing equal proportions of R2 and either R1 or R3. D, one R2 monomer is not sufficient to maintain R2 regulatory dominance in a heterotetramer containing three R1 subunits.