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.
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.
Authors: Huma Saleem; Stephen C Tovey; Taufiq Rahman; Andrew M Riley; Barry V L Potter; Colin W Taylor Journal: PLoS One Date: 2013-01-25 Impact factor: 3.240