Differential codes for free Ca2+-calmodulin signals in nucleus and cytosol

Background: Many targets of calcium signaling pathways are activated or inhibited by binding the Ca2+-liganded form of calmodulin (Ca2+-CaM). Here, we test the hypothesis that local Ca2+-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca2+-CaM concentration. Results: Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca2+-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca2+-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. in contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca2+-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca2+-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca2+-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca2+-CaM-binding protein at high concentration in the cytosol. Conclusions: Subcellular differences in the distribution of Ca2+-CaM-binding proteins can produce gradients of free Ca2+-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca2+-CaM concentrations, and thus the local activity of Ca2+-CaM targets. Free Ca2+-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca2+-CaM.