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

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 (IP₃R) was selected as the target protein and a malachite green-conjugated IP₃ analog, MGIP₃, was used as a small-molecular probe. We examined the effect of MGIP₃-based CALI on Ca²⁺ release via IP₃R using permeabilized smooth muscle cells. When the cells were treated with MGIP₃ followed by laser irradiation, the IP₃-induced Ca²⁺ release rate was decreased in a concentration- and irradiation time-dependent manner. The effect was specific for IP₃R, because the Ca²⁺ uptake function of the co-localized sarco/endoplasmic reticulum Ca²⁺-ATPase was not affected. Conclusions: IP₃R was specifically inactivated by smCALI using MGIP₃. The efficiency of inactivation was calculated to be substantially greater than that of antibody-based CALI. The efficient and specific inactivation of IP₃R 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 for a wider application of smCALI as a useful method to study spatiotemporal protein functions.