A fluorescence-based method for evaluating inositol 1,4,5-trisphosphate receptor ligands: determination of subtype selectivity and partial agonist effects.

Inositol 1,4,5-trisphosphate (IP₃) receptors consist of three subtypes: IP₃R1, IP₃R2, and IP₃R3. Although numerous IP₃ receptor ligands have been synthesized, none of the subtype-selective ligands are known. We have developed a simple fluorescence method to examine the subtype selectivity of IP₃ receptor ligands using FRET-based IP₃ biosensors LIBRAvI, LIBRAvII, and LIBRAvIII. The addition of IP₃ or adenophostin A (ADA) to permeabilized biosensor-expressing cells increased the fluorescence ratios of these biosensors in a concentration-dependent manner, and the potency of ADA relative to that of IP₃ in terms of the changes in the fluorescence ratios of LIBRAvI, LIBRAvII, and LIBRAvIII was 43-, 22-, and 28-fold, respectively. This fluorescence-based method further showed that several ADA analogs had significant differences with respect to subtype selectivity and potency. These results highlight the important role played by the O-glycosidic structure of ADA in the selectivity of the ligands for IP₃R1, as evidenced by the modified selectivity following replacement of the 5'-hydroxyl with a phenyl or phenethyl group. We also found that one ADA analog 5'-deoxy-5'-phenyladenophostin A possessed a partial agonistic effect on IP₃R1. Together, the novel fluorescent methods described herein are useful for the evaluation of properties of IP₃R ligands, including potency, efficacy, and subtype selectivity.