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

Abstract

Background: Chromophore-assisted laser inactivation (CALI) is a powerful method for the study of in situ protein function in cellular processes. By using CALI, it is possible to abrogate the function of a target protein with unprecedented spatial and temporal resolution. However, CALI has some limitations, which restrict wider biological application, owing mainly to the use of antibody for target recognition. To circumvent the limitations, we have developed small molecule-based CALI (smCALI). Results: The inositol 1,4,5-trisphosphate receptor (IP 3R) was selected as the target protein and a malachite green-conjugated IP 3 analog, MGIP 3, was used as a small-molecular probe. We examined the effect of MGIP 3-based CALI on Ca 2+ release via IP 3R using permeabilized smooth muscle cells. When the cells were treated with MGIP 3 followed by laser irradiation, the IP 3-induced Ca 2+ release rate was decreased in a concentration- and irradiation time-dependent manner. The effect was specific for IP 3R, because the Ca 2+ uptake function of the co-localized sarco/endoplasmic reticulum Ca 2+-ATPase was not affected. Conclusions: IP 3R 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 IP 3R in various cell functions. Our results may be considered to be a first step for a wider application of smCALI as a useful method to study spatiotemporal protein functions.