Characterizing the response of calcium signal transducers to generated calcium transients

Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological p henomena as diverse as muscle contraction, neurosecretion, and cell divisio n. When Ca2+ is rapidly mixed with slow Ca2+ chelators, EGTA, or Mg2+/EDTA, artificial Ca2+ transients (ACTs) of varying duration (0.1-50 ms half-widt hs (hws)) and amplitude can be generated. We have exposed several Ca2+ indi cators, Ca2+-binding proteins, and a Ca2+-dependent enzyme to ACTs of vario us durations and observed their transient binding of Ca2+, complex formatio n, and/or activation. A 0.1 ms hw ACT transiently occupied similar to 70% o f the N-terminal regulatory sites of troponin C consistent with their rapid Ca2+ on-rate (8.7 +/- 2.0 x 10(7) M-1 s(-1)). A 1.1 ms hw ACT produced sim ilar to 90% transient binding of the N-terminal of calmodulin (CaM) to the RS-20 peptide, but little binding of CaM's C-terninal to RS-20. A 0.6 ms hw ACT was sufficient fur the N-terminal of CaM to transiently bind similar t o 60% of myosin light chain kinase (MLCK), while a 1.8 ms hw ACT produced s imilar to 22% transient activation of the sarcoplasmic reticulum (SR) Ca2+/ ATPase, Ln both cases, the ACT had fallen back to baseline similar to 10-30 ms before maximal binding of CaM to MLCK or SR Ca2+/ATPase activation occu rred and binding and enzyme activation persisted long after the Ca transien t had subsided. The use of ACTs has allowed us to visualize how the Ca2+-ex change rates of Ca2+-binding proteins dictate their Ca2+-induced conformati onal changes, Ca2+-induced protein/peptide and protein/protein interactions , and enzyme activation and inactivation, in response to Ca2+ transients of various amplitude and duration. By characterizing the response of these pr oteins to ACTs, we can predict with greater certainty how they would respon d to natural Ca2+ transients to regulate cellular phenomena.