Characterizing the response of calcium signal transducers to generated calcium transients.
Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological phenomena as diverse as muscle contraction, neurosecretion, and cell division. 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-widths (hws)) and amplitude can be generated. We have exposed several Ca2+ indicators, Ca2+-binding proteins, and a Ca2+-dependent enzyme to ACTs of various durations and observed their transient binding of Ca2+, complex formation, and/or activation. A 0.1 ms hw ACT transiently occupied approximately 70% of 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 approximately 90% transient binding of the N-terminal of calmodulin (CaM) to the RS-20 peptide, but little binding of CaM's C-terminal to RS-20. A 0.6 ms hw ACT was sufficient for the N-terminal of CaM to transiently bind approximately 60% of myosin light chain kinase (MLCK), while a 1.8 ms hw ACT produced approximately 22% transient activation of the sarcoplasmic reticulum (SR) Ca2+/ATPase. In both cases, the ACT had fallen back to baseline approximately 10-30 ms before maximal binding of CaM to MLCK or SR Ca2+/ATPase activation occurred and binding and enzyme activation persisted long after the Ca transient had subsided. The use of ACTs has allowed us to visualize how the Ca2+-exchange rates of Ca2+-binding proteins dictate their Ca2+-induced conformational 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 proteins to ACTs, we can predict with greater certainty how they would respond to natural Ca2+ transients to regulate cellular phenomena.
Davis
J P
JP
Department of Medical Biochemistry, The Ohio State University Medical Center, Columbus 43210, USA.
Tikunova
S B
SB
Walsh
M P
MP
Johnson
J D
JD
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000634:Aminoquinolines / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000378:metabolism
D000634:Aminoquinolines; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019209:Troponin C; C061167:RS 20; D004533:Egtazic Acid; D009219:Myosin-Light-Chain Kinase; D000251:Adenosine Triphosphatases; C035739:Quin2; D002118:Calcium