Small molecule-based laser inactivation of inositol 1,4,5-trisphosphate receptor

Background: Chromophore-assisted laser inactivation (CALI) is a powerful me thod for the study of in situ protein function in cellular processes. By us ing CALI, it is possible to abrogate the function of a target protein with unprecedented spatial and temporal resolution. However. CALI has some limit ations, which restrict wider biological application, owing mainly to the us e of antibody for target recognition. To circumvent the limitations, we hav e developed small molecule-based CALI (smCALI). Results: The inositol 1,4.5-trisphosyhate receptor (IP3R) was selected as t he target protein and a malachite green-conjugated IF? analog. MGIP(3), was used as a small-molecular probe. We examined the effect of MGIP(3)-based C ALI on Ca2+ Irelease via IP3R using permeabilized smooth muscle cells. When the cells were treated with MGIP3 Followed by laser irradiation, the IP3-i nduced Ca2+ release rate was decreased in a concentration- and irradiation time-dependenr manner. The effect was specific for IP3R. because the Ca2+ u ptake function of the co-localized sarco/endoplasmic reticulum Ca2+-ATPase was not affected was not affected. Conclusions: IP3R was specifically inactivated by smCALI using MGIP(3). The efficiency of inactivation was calculated to be substantially greater than that of antibody-based CALI. The efficient and specific inactivation of IP 3R would allow us to obtain an insight into spatiotemporal roles of IP3R in various cell functions. Our results may be considered to be a first step f or a wider application of smCALI as a useful method to study spatiotemporal protein functions. (C) 2001 Elsevier Science Ltd. All rights reserved.