Translational Mobility of the Type 3 Inositol 1,4,5-Trisphosphate Receptor Ca2+Release Channel in Endoplasmic Reticulum Membrane

Michelle Ferreri-Jacobia, Don-On Daniel Mak, J. Kevin Foskett
2004 Journal of Biological Chemistry  
The inositol 1,4,5-trisphosphate receptor (InsP 3 R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca 2؉ release channel. InsP 3 -mediated Ca 2؉ release modulates the cytoplasmic free Ca 2؉ concentration ([Ca 2؉ ] i ), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP 3 R is believed to be important for providing local [Ca 2؉ ] regulation and for ensuring efficient
more » ... nal coupling between Ca 2؉ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca 2؉ -induced Ca 2؉ release. Highly localized Ca 2؉ release has been attributed to the ability of the InsP 3 R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP 3 R (InsP 3 R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP 3 R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was ϳ4 ؋ 10 ؊10 cm 2 /s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (ϳ75%) of channels mobile. D was modestly increased at 37°C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP 3 R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP 3 R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATPand microtubule-facilitated motility of the channel. 1 The abbreviations used are: InsP 3, inositol 1,4,5-trisphosphate; [Ca 2ϩ ] i , cytoplasmic free Ca 2ϩ concentration; CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; InsP 3 R, inositol 1,4,5-trisphosphate receptor; PBS, phosphate-buffered saline.
doi:10.1074/jbc.m409462200 pmid:15537642 fatcat:gwbuq5e5gbgenizlpo3wm5fxgq