BINDING OF H-3 MELATONIN TO CALMODULIN

Studies in melatonin mechanism of action have suggested that one of th em could be the binding of the hormone to calmodulin. We assessed calm odulin-melatonin binding by combining liposome incorporation of calmod ulin with sepa ration of free and bound H-3-Melatonin by a rapid ultra filtration method. Specific binding to calmodulin was saturable, reve rsible, Ca++-dependent, ligand selective, and showed high affinity. Sa turation as well as associa tion-dissociation studies revealed that H- 3-Melatonin binds to a single site on the calmodulin molecule with a K d of 188 pM and a total binding capacity Bmax of 35 pM/ug of calmoduli n. Displacement experiments showed that the relative order of potency of some compounds for inhibition of H-3-Melatonin was as follows: Mela tonin > 6-chloromela tonin > 6-hydroxymelatonin > luzindole > trifluop erazine. The results explain our previously reported melatonin effects such as cytoskeletal rearrangements, inhibition of calmodulin depende nt phosphodiesterase activity as well as the modification of Ca++-calm odulin electrophoretic mobility. The high affinity of melatonin bindin g to calmodulin suggests that the hormone is able to modulate cell act ivity by intracellularly binding to calmodulin at physiologically rang es. Melatonin-calmodulin binding could modulate many intracellular Ca+ functions and thus, the set-point for cell activity will follow the rhythmic circulating levels of the pineal hormone. Moreover, since cal modulin and melatonin are phylogenetically well preserved compounds, t heir interaction may represent a primary mechanism for both the regula tion and the synchronization of cell physiology.