CONFORMATIONAL-CHANGES IN PLANT INS(1,4,5)P-3 RECEPTOR ON INTERACTIONWITH DIFFERENT MYOINOSITOL TRISPHOSPHATES AND ITS EFFECT ON CA2+ RELEASE FROM MICROSOMAL FRACTION AND LIPOSOMES

The interaction of the only reported plant inositol trisphosphate rece ptor with different myo-inositol trisphosphates (InsP(3) species), nam ely Ins(1,4,5)P-3, Ins(1,3,4)P-3, Ins(1,5,6)P-3 and Ins(2,4,5)P-3, wer e studied to assess the extent of Ca2+ mobilization from microsomes/va cuoles as well as liposomes in vitro. Ins(1,4,5)P-3 and Ins(2,4,5)P-3 bind with the receptor with comparable affinities, as evidenced from t heir dissociation constants (K-d approx. 100 nM at 5 degrees C), where as the interaction between Ins(1,3,4)P-3/Ins(1,5,6)P-3 and the recepto r was not detected even with these ligands at 5 mu M. Ins(1,3,4)P-3/In s(1,5,6)P-3 isomers also do not elicit Ca2+ release from liposomes or microsomes/vacuoles. The ability of any InsP(3) to bind the receptor f or Ins(1,4,5)P-3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not hel p to correlate it directly with the extent of Ca2+ release from the in tracellular stores, because the concentration of Ca2+ released by Ins( 1,4,5)P-3 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)P-3 under id entical conditions. To understand the role of the receptor conformatio n in Ca2+ release by different isomers, we have probed the conformatio nal change of the receptor when the different isomers bind to it. Acce ssibility of the tryptophan residues in the free and Ins(1,4,5)P-3/Ins (2,4,5)P-3-bound receptor was monitored by a neutral fluorescence quen cher, acrylamide. The resulting Stern-Volmer-type quenching plots of t he internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P-3 and Ins(2,4,5)P-3. It is also det ected when far-UV CD spectra (205-250 nm) of the free and ligand [Ins( 1,4,5)P-3/Ins(2,4,5)P-3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational cha nges induced by the two isomers are different in nature. When thermody namic parameters, such as enthalpy (Delta H), entropy (Delta S) and fr ee energy (Delta G), for the formation of the two InsP(3)-receptor com plexes are compared, a major difference in the extent of changes in en thalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the req uisite conformational change in the receptor that determines the poten cy of the InsP(3) isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.