Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane

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 Ca2+ release channel. InsP(3)-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+](i)), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP,R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R 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 similar to4 x 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 (similar to75%) of channels mobile. D was modestly increased at 37 degreesC, 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.