A key assumption of most models for calmodulin regulation of smooth and non-muscle contractility is that calmodulin is freely diffusible at resting intracellular concentrations of free Ca. However, fluorescence recovery after photobleaching (FRAP) measurements of three different fluorescent analogs of calmodulin in cultured bovine tracheal smooth muscle cells suggest that free calmodulin may be limiting in unstimulated cells. Thirty-seven % of microinjected calmodulin is immobile by FRAP and the fastest recovering component has an effective diffusion coefficient 7-fold slower than a dextran of equivalent size. Combining the FRAP data with extraction data reported in a previous paper (Tansey, M., Luby-Phelps, K., Kamm, K. E., and Stull, J. T.(1994) J. Biol. Chem. 269, 9912-9920), we estimate that at most 5% of total endogenous calmodulin in resting smooth muscle cells is unbound (freely diffusible). Examination of the Ca dependence of calmodulin mobility in permeabilized cells reveals that binding persists even at intracellular Ca concentrations as low as 17 nM. When Ca is elevated to between 450 nM and 3 µM, some of the bound calmodulin is released, as indicated by an increase in the effective diffusion coefficient and the percent mobile fraction. At higher Ca, calmodulin becomes increasingly immobilized. In about 50% of the cell population, clamping Ca at micromolar levels results in translocation of cytoplasmic calmodulin to the nucleus. The compartmentalization and complex dynamics of calmodulin in living smooth muscle cells have profound implications for understanding how calmodulin regulates contractility in response to extracellular signals.

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