CA2-REGULATED DYNAMIC COMPARTMENTALIZATION OF CALMODULIN IN LIVING SMOOTH-MUSCLE CELLS()

A key assumption of most models for calmodulin regulation of smooth an d non-muscle contractility is that calmodulin is freely diffusible at resting intracellular concentrations of free Ca2+. However, fluorescen ce recovery after photobleaching (FRAP) measurements of three differen t fluorescent analogs of calmodulin in cultured bovine tracheal smooth muscle cells suggest that free calmodulin may be limiting in unstimul ated cells, Thirty-seven % of microinjected calmodulin is immobile by FRAP and the fastest recovering component has an effective diffusion c oefficient 7-fold slower than a dextran of equivalent size. Combining the FRAP data with extraction data reported in a previous paper (Tanse y, 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 diffusib le). Examination of the Ca2+ dependence of calmodulin mobility in perm eabilized cells reveals that binding persists even at intracellular Ca 2+ concentrations as low as 17 nM. When Ca2+ is elevated to between 45 0 nM and 3 mu M, some of the bound calmodulin is released, as indicate d by an increase in the effective diffusion coefficient and the per ce nt mobile fraction, At higher Ca2+, calmodulin becomes increasingly im mobilized. In about 50% of the cell population, clamping Ca2+ at micro molar 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 understan ding how calmodulin regulates contractility in response to extracellul ar signals.