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Calbindin D28k Exhibits Properties Characteristic of a Ca2+ Sensor

2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês

10.1074/jbc.m200415200

1083-351X

Tord Berggård, Simona Miron, Patrik Önnerfjord, Eva Thulin, Karin S. Åkerfeldt, Jan J. Enghild, Mikael Akke, Sara Linse,

Environmental Toxicology and Ecotoxicology

Calbindin D 28k is a member of the calmodulin superfamily of Ca 2+ -binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca 2+ buffer, but the studies presented in this work indicate that it may also act as a Ca 2+ sensor. The results show that Mg 2+ binds to the same sites as Ca 2+ with an association constant of ∼1.4·10 3 m −1 in 0.15 m KCl. The four high affinity sites in calbindin D 28k bind Ca 2+ in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg 2+ , the Ca 2+ affinity is reduced by a factor of 2, and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca 2+ concentration in a resting cell calbindin D 28k is saturated to 40–75% with Mg 2+ but to less than 9% with Ca 2+ . In contrast, the protein is expected to be nearly fully saturated with Ca 2+ at the Ca 2+ level of an activated cell. A substantial conformational change is observed upon Ca 2+ binding, but only minor structural changes take place upon Mg 2+ binding. This suggests that calbindin D 28k undergoes Ca 2+ -induced structural changes upon Ca 2+ activation of a cell. Thus, calbindin D 28k displays several properties that would be expected for a protein involved in Ca 2+ -induced signal transmission and hence may function not only as a Ca 2+ buffer but also as a Ca 2+ sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca 2+ , becomes exposed upon Ca 2+ binding.