Mechanism of Ca2+-dependent nuclear accumulation of calmodulin

The intracellular Ca2+ receptor calmodulin (CaM) coordinates responses to e xtracellular stimuli by modulating the activities of its various binding pr oteins. Recent reports suggest that, in addition to its familiar functions in the cytoplasm, CaM may be directly involved in rapid signaling between c ytoplasm and nucleus. Here we show that Ca2+ dependent nuclear accumulation of CaM can be reconstituted in -permeabilized cells. Accumulation was bloc ked by M13, a CaM antagonist peptide, but did not require cytosolic factors or an ATP regenerating system. Ca2+-dependent influx of CaM into nuclei wa s not blocked by inhibitors of nuclear localization signal-mediated nuclear import in either permeabilized or intact cells, Fluorescence recovery afte r photobleaching studies of CaM in intact cells showed that influx is a fir st-order process with a rate constant similar to that of a freely diffusibl e control molecule (20-kDa dextran). Studies of CaM efflux from preloaded n uclei in permeablized cells revealed the existence of three classes of nucl ear binding sites that are distinguished by their Ca2+-dependence and affin ity. At high [Ca2+], efflux was enhanced by addition of a high affinity CaM -binding protein outside the nucleus. These data suggest that CaM diffuses freely through nuclear pores and that CaM-binding proteins in the-nucleus a ct as a sink for Ca2+-CaM, resulting in accumulation of CaM in the nucleus on elevation of intracellular free Ca2+.