We used quick‐freeze deep‐etch replica electron microscopy to visualize the native structure of inositol‐1,4,5‐trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two‐dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square‐shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row‐line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule.
Abstract:To identify which organelles contained inositol trisphosphate (InsP 3 ) receptor type 2 (InsP 3 R2) in adrenal medullary (AM) cells, immunocytochemical and biochemical studies were performed on AM cells of several species. InsP 3 R2-like immunoreactive materials produced by two different anti-InsP 3 R2 antibodies (Abs) (Chemicon and Sigma) were distributed in rat AM cells in agreement with BODIPY-FL-InsP 3 binding sites. For two other Abs (KM1083 and Santa Cruz), some of the antiInsP 3 R2 immunoreactive materials were stained with an antidopamine-β-hydroxylase Ab, but not by BODIPY-FL-InsP 3 . BO-DIPY-FL-thapsigargin binding sites were consistent with a distribution of the endoplasmic reticulum (ER) identified by an anticalnexin Ab, and a prior application of thapsigargin significantly eliminated BODIPY-FL-thapsigargin bindings, suggesting that BODIPY-FL-thapsigargin bindings were mediated by thapsigargin, but not the fluorescence molecule. The anti-InsP 3 R2 Ab that produced stainings consistent with BODIPY-FL-InsP 3 bindings recognized a protein with about 250 kDa. A fractional analysis of bovine adrenal medullae revealed that the 250 kDa InsP 3 R2 was detected in a crude membrane fraction, but not in a secretory granule fraction. The results suggest that the InsP 3 R2 was present in the ER, but not in secretory granules in AM cells.
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