Three-dimensional structure of recombinant type 1 inositol 1,4,5-trisphosphate receptor

Abstract

IP3Rs (inositol 1,4,5-trisphosphate receptors) are the intracellular channels that mediate release of Ca2+ from the endoplasmic reticulum in response to the many stimuli that evoke Ins(1,4,5)P3 formation. We characterized and purified type 1 IP3R heterologously expressed in Sf9 insect cells, and used the purified IP3R1 to determine its three-dimensional structure by electron microscopy and single-particle analysis. Recombinant IP3R1 has 4-fold symmetry with overall dimensions of approx. 19.5 nm x 19.5 nm x 17.5 nm. It comprises a small domain, which is likely to include the pore, linked by slender bridges to a large cytoplasmic domain with four petal-like regions. Our structures of recombinant IP3R1 and native cerebellar IP3R have similar appearances and dimensions. The only notable difference is the absence of a central stigma-like domain from the cytoplasmic region of recombinant IP3R1. The first structure of a recombinant IP3R is an important step towards developing three-dimensional structures of IP3R that better contribute to our understanding of the structural basis of IP3R activation.

Figure 1. Recombinant IP₃R1 is functional

(A) The recombinant IP₃R1 used has an N-terminal ECFP tag, a PreScission protease cleavage site (Pr) and the biotinylation sequence (biotin). (B) Ins(1,4,5)P₃-evoked Ca²⁺ release from permeabilized Sf9 cells with or without expression of recombinant IP₃R1. (C) Specific binding of [³H]Ins(1,4,5)P₃ (1.5 nM) in the presence of the indicated concentrations of Ins(1,4,5)P₃ (IP₃) to membranes prepared from Sf9 cells expressing recombinant IP₃R1. Results (B and C) are means±S.E.M (n=3).

Figure 2. Purification of recombinant IP₃R

(A and B) Silver-stained gel (A, 0.12 μg of protein/lane) and immunoblot with an IP₃R1-specific antiserum (B, 0.12 μg of protein/lane) of purified recombinant IP₃R1 from fractions 20–24 of the gel-filtration step. Molecular-masses are shown in kDa. Results are typical of at least five similar analyses. (C) Specific binding of [³H]Ins(1,4,5)P₃ (1.5 nM) in the presence of the indicated concentrations of Ins(1,4,5)P₃ to purified recombinant IP₃R1 (means±S.E.M, n=3). (D) Electron micrograph of purified recombinant IP₃R1 highlighting particles (arrows) with the expected size of tetrameric IP₃R (~20 nm diameter). Scale bar, 70 nm.

Figure 3. Image analysis of IP₃R particles

(A) Eigenimages of the unaligned dataset. Eigenimage 1 shows the sum of all images. Eigenimages 3 and 4 show approx. 3-fold symmetry. Eigenimages 5 and 6 show approx. 2-fold symmetry. Eigenimages 7 and 8 show 4-fold symmetry. (B) Class averages obtained independently after two iterative alignments (reference-free classes). (C) Similar class averages obtained after three iterative alignments using the filtered three-dimensional structure of native IP₃R [23] as a reference for the first alignment. (D) Class averages used for the final three-dimensional reconstruction. (E) Re-projections of the final three-dimensional reconstruction corresponding to the class averages shown in (D). (F) Surface views of the final three-dimensional reconstruction in the same orientations as the class averages. Scale bars (A–F), 20 nm.

Figure 4. Structure of recombinant IP₃R1

(A) Each section (1–40) is 0.45 nm thick and viewed along the symmetry axis starting from the cytoplasmic end (1) to the end likely to be within the ER lumen (40). Scale bar, 10 nm. (B) Surface views of the three-dimensional reconstruction of recombinant IP₃R1 viewed from the cytosol (i), from the lumen of the ER (ii) and two views in cross-section (iii and iv). (C) Views similar to those shown in (B), but for native cerebellar IP₃R [23]. Scale bars (B and C), 20 nm.