title: Conformational changes in plant Ins(1,4,5)P3 receptor on interaction with different myo-inositol trisphosphates and its effect on Ca2+ release from microsomal fraction and liposomes.
abstract: The interaction of the only reported plant inositol trisphosphate receptor with different myo-inositol trisphosphates (InsP3 species), namely Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,5,6)P3, and Ins(2,4,5)P3, were studied to assess the extent of Ca2+ mobilization from microsomes/vacuoles as well as liposomes in vitro. Ins(1,4,5)P3 and Ins(2,4,5)P3 bind with the receptor with comparable affinities, as evidenced from their dissociation constants (Kd approx. 100 nM at 5 degrees C), whereas the interaction between Ins(1,3,4)P3/Ins(1,5,6)P3 and the receptor was not detected even with these ligands at 5 microM. Ins(1,3,4)P3/Ins(1,5,6)P3 isomers also do not elicit Ca2+ release from liposomes or microsomes/ vacuoles. The ability of any InsP3 to bind the receptor for Ins(1,4,5)P3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not help to correlate it directly with the extent of Ca2+ release from the intracellular stores because the concentration of Ca2+ released by Ins(1,4,5)P3 as estimated over a period of 20 s is 3500 +/- 200 nM/mg of protein and is about 4-fold higher than that by Ins(2,4,5)P3 under identical conditions. To understand the role of the receptor conformation in Ca2+ release by different isomers, we have probed the conformational change of the receptor when the different isomers bind to it. Accessibility of the tryptophan residues in the free and Ins(1,4,5)P3/Ins(2,4,5)P3-bound receptor was monitored by a neutral fluorescence quencher, acrylamide. The resulting Stern-Volmer-type quenching plots of the internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P3 and Ins(2,4,5)P3. It is also detected when far-UV CD spectra (205-250 nm) of the free and ligand [Ins(1,4,5)P3/Ins(2,4,5)P3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational changes induced by the two isomers are different in nature. When thermodynamic parameters, such as enthalpy (delta H), entropy (delta S) and free energy (delta G), for the formation of the two InsP3-receptor complexes are compared, a major difference in the extent of changes in enthalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the requisite conformational change in the receptor that determines the potency of the InsP3 isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.
authors.affiliation: Department of Biophysics, Molecular Biology and Genetics, University of Calcutta, India.
authors.forename: S
authors.initials: S
authors.lastname: Dasgupta
authors.forename: D
authors.initials: D
authors.lastname: Dasgupta
authors.forename: A
authors.initials: A
authors.lastname: Chatterjee
authors.forename: S
authors.initials: S
authors.lastname: Biswas
authors.forename: B B
authors.initials: BB
authors.lastname: Biswas
mesh_terms: D020106:Acrylamide; D000178:Acrylamides; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D007887:Fabaceae / Q000737:chemistry*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007424:Intracellular Fluid / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D010946:Plants, Medicinal; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013816:Thermodynamics
chemical_list: D000178:Acrylamides; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008081:Liposomes; D018160:Receptors, Cytoplasmic and Nuclear; D020106:Acrylamide; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium