Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits

Abstract

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

Keywords: ATP; Calcium Imaging; Calcium Intracellular Release; Calcium Signaling; Inositol 1,4,5-Trisphosphate Receptor; Signal Transduction; Single Channel Recording.

FIGURE 1.

Differential expression and the formation of homo- and heteromeric IP₃R complexes in native tissues. A, lysates from the indicated mouse tissues were prepared, and equivalent amounts of proteins were resolved on SDS-polyacrylamide gels, transferred to a nitrocellulose, and processed for immunoblots with the indicated antibodies. Lysates from DT40–3KO cells (3KO) expressing IP₃R1 (R1), IP₃R2 (R2), or IP₃R3 (R3) were used as controls. B, lysates from the indicated mouse tissues were prepared as in A. IP₃Rs were immunoprecipitated with subtype-specific antibodies. Immunoprecipitates were processed for immunoblots with the indicated antibodies. C, lysates from mouse pancreas were prepared and subjected to sequential immunoprecipitation first with anti-IP₃R2 followed by anti-IP₃R1 to deplete R2 (middle lane) and both R1 and R2 (right lane) as described under “Materials and Methods.” Equivalent supernatants, before and after immunodepletion (Id), were fractionated on SDS-PAGE and probed with the indicated antibodies. Shown is a representative experiment. D, histograms comparing R3 immunoreactivities after immunodepletion of R2 (middle column) or R1 and R2 (right column). R3 immunoreactivities were quantified and expressed as percentage of immunoreactivity before immunodepletion. Data are presented as mean ± S.E.

FIGURE 2.

Construction of concatenated IP₃Rs and generation of stable DT40–3KO cell lines. A, schematic representation of pJAZZmamm showing its main structural features as follows: left arm contains the plasmid origin of replication, a transcriptional terminator, CMV promoter, and NcoI site; right arm, cut with SwaI to result in a blunt end, contains a transcriptional terminator and bacterial and mammalian antibiotic resistance genes. The inset contains a tandem construct of IP₃R1 head cDNA digested with NcoI and AgeI and an IP₃R1 tail cDNA digested with AgeI and HpaI (blunt end). B, DT40–3KO cells stably expressing various IP₃R constructs were harvested and lysed, and ∼60 μg of lysate proteins were fractionated on 4% gels and processed in immunoblots with either anti-IP₃R1 antibody (B) or anti-IP₃R2 antibody (C). Only 15 μg of lysates were loaded for R1 and R2 lanes because R1 and R2 are very much overexpressed. D, cell lysates were prepared from DT40–3KO cells expressing either R1 or increasingly longer concatenated constructs as follows: R1R1, R1R1R1, or R1R1R1R1. Proteins were fractionated on 3–5% gradient gels, transferred to nitrocellulose, and immunoblotted with anti-IP₃R1. Less lysate proteins (15 μg) were loaded in the R1 lane relative to other lanes (60 μg). Asterisks denote nonspecific bands.

FIGURE 3.

Targeting of dimeric IP₃R into ER membranes. DT40–3KO cells stably expressing R1 (A) or R1R1 (B) were harvested and lysed (lysates) or homogenized, and cytosol and membrane fractions were prepared by ultracentrifugation. Equivalent amounts of proteins were fractionated and processed in immunoblots with anti-IP₃R1 (upper panels), anti-SERCA (middle panels), and anti-GAPDH (lower panels). Representative experiments are shown.

FIGURE 4.

Dimeric IP₃Rs migrate as tetrameric complexes on native gels. Lysates from DT40–3KO cells stably expressing various IP₃R constructs were prepared using CHAPS lysis buffer. Lysates were then resolved on 3–12% NativePAGE^TM Novex. Proteins were immunoblotted with anti-IP₃R1 (A) or anti-IP₃R2 (B). Representative experiments are shown. C, cell lysates were prepared from R1 and R1R1 cells and subjected to ultracentrifugation. Cleared supernatants were injected through a prepacked HiPrep 16/60 Sephacryl S-400 HR gel filtration column. Fractions of eluates were collected (1 ml/sample) and were separated on SDS-PAGE and processed by immunoblotting. IP₃R1 immunoreactivities were quantified and expressed as percentage of the peak immunoreactivity.

FIGURE 5.

Robust IP₃-mediated Ca²⁺ release activity in cells expressing dimeric IP₃R constructs. In A, DT40–3KO (3KO) or 3KO stably expressing various IP₃R constructs were loaded with Fura-2AM and stimulated with 500 nm trypsin to induce IP₃ formation. Ca²⁺ release was measured as a normalized change in the 340/380 fluorescence ratio to facilitate the comparison of fold changes in the various cell lines. B, histograms depict the % of cells expressing the indicated constructs that responded to trypsin with >0.1 change in the 340/380 fluorescence ratio. C, average maximum increase over basal 340/380 fluorescence ratio after trypsin stimulation in cells expressing the constructs indicated. Experiments were repeated at least four times with greater than 30 cells analyzed in each experiment. Data are presented as mean ± S.E.

FIGURE 6.

The presence of IP₃R2 in the assembled channel results in Ca²⁺ oscillations. DT40–3KO stably expressing various IP₃R constructs were loaded with Fura-2AM and stimulated with 1 μg/ml anti-chicken IgM. Ca²⁺ release was measured as a change in the 340/380 fluorescence ratio. Shown are representative Ca²⁺ traces from DT40–3KO stably expressing R1 (A), R1R1 (B), R1R2 (C), R2R1 (D), R2R2 (E), and R2 (F). G, the histogram shows the % of cells expressing the indicated construct responding to 1 μg/ml IgM (numbers in parentheses indicate the total number of cells/number of experimental runs). H, % of responding cells exhibiting oscillatory behavior for each construct is shown. Oscillating cells were defined as cells with three or more peaks following IgM exposure.

FIGURE 7.

Single channel recording of R1 and R1R1 channels. Single channel recordings were conducted by on-nucleus configuration of the patch clamp technique. A, representative single channel records from monomeric R1 or from dimeric R1R1 stimulated with 1 or 10 μm IP₃ in the presence of 200 nm free Ca²⁺ and 5 mm ATP. B, histograms depict channel open probabilities for both R1 and R1R1 activated by 1 or 10 μm IP₃. C, current versus voltage relationship for R1 and R1R1 channels.

FIGURE 8.

Single channel recording of homo- and heteromeric channels. Single channel recordings were conducted as in Fig. 7. A, single channel recordings under the indicated conditions, representing “high” and “low” [ATP] from DT40–3KO cells stably expressing R1R1 dimeric construct. The modulation of activity by ATP is characteristic of the R1. B, recordings under the indicated conditions from DT40–3KO cells stably expressing R2R2 dimeric construct. The modulation of activity by ATP is characteristic of R2. C, recordings under the indicated conditions from DT40–3KO cells stably expressing R1R2 dimeric construct. The modulation of activity by ATP is characteristic of the R2. D, pooled data for the indicated conditions. Data from R1 and R2 are modified from Ref. .

FIGURE 9.

Analyses of IP₃R channel kinetics. Single channel recordings were obtained under the indicated conditions as in Figs. 7 and 8. Burst length and interburst intervals were calculated as described under “Materials and Methods.” Shown are histograms representing burst length time constants (τ) (black bar) and interburst interval time constants (red bars) for the indicated [ATP] and [IP₃], for R1R1 (A and B), R2R2 (C and D), and R1R2 (E and F).