Dynamics of the Ins(1,4,5)P3 receptor during polarization of MDCK cells

Abstract

The uneven distribution of the Ins(1,4,5)P3R [Ins(1,4,5)P3 receptor] within the ER (endoplasmic reticulum) membrane generates spatially complex Ca2+ signals. The ER is a dynamic network, which allows the rapid diffusion of membrane proteins from one part of the cell to another. However, little is known about the localization and the dynamics of the Ins(1,4,5)P3R in the ER of living cells. We have used a MDCK (Madin-Darby canine kidney) clone stably expressing the Ins(1,4,5)P3R1-GFP (where GFP stands for green fluorescent protein) to investigate the effect of cell polarity on the lateral mobility of the Ins(1,4,5)P3R. In non-confluent MDCK cells, the chimaera is homogeneously distributed throughout the ER and the nuclear envelope. FRAP (fluorescence recovery after photobleaching) experiments showed that the receptor can move freely in the ER with a diffusion constant (D=0.01 microm2/s) approx. ten times lower than other ER membrane proteins. In confluent polarized cells, two populations of receptor can be defined: one population is distributed in the cytoplasm and is mobile but with a slower diffusion constant (D=0.004 microm2/s) compared with non-confluent cells, whereas the other population is concentrated at the periphery of the cells and is apparently immobile. The observed differences in the mobility of the Ins(1,4,5)P3R are most probably due to its interactions with stable protein complexes that form at the periphery of the polarized cells.