title: Ca2+ binding effects on protein conformation and protein interactions of canine cardiac calsequestrin.
abstract: Calsequestrin is a Ca2+-binding protein located intraluminally in the junctional sarcoplasmic reticulum (SR) of striated muscle. In this study, Ca2+ binding to cardiac calsequestrin was assessed directly by equilibrium dialysis and correlated with effects on protein conformation and calsequestrin's ability to interact with other SR proteins. Cardiac calsequestrin bound 800-900 nmol of Ca2+/mg of protein (35-40 mol of Ca2+/mol of calsequestrin). Associated with Ca2+ binding to cardiac calsequestrin was a loss in protein hydrophobicity, as revealed with use of absorbance difference spectroscopy, fluorescence emission spectroscopy, and photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125]iodophenyl)diazirine. Ca2+ binding to cardiac calsequestrin also caused a large change in its hydrodynamic character, almost doubling the sedimentation coefficient. We observed that cardiac calsequestrin was very resistant to several proteases after binding Ca2+, consistent with a global effect of Ca2+ on protein conformation. Moreover, Ca2+ binding to cardiac calsequestrin completely prevented its interaction with several calsequestrin-binding proteins, which we identified in cardiac junctional SR vesicles for the first time. The principal calsequestrin-binding protein identified in junctional SR vesicles exhibited an apparent Mr of 26,000 in sodium dodecyl sulfate-polyacrylamide gels. This 26-kDa calsequestrin-binding protein was greatly reduced in free SR vesicles and absent from sarcolemmal vesicles and was different from phospholamban, an SR regulatory protein exhibiting a similar molecular weight. Our results suggest that the specific interaction of calsequestrin with this 26-kDa protein may be regulated by Ca2+ concentration in intact cardiac muscle, when the Ca2+ concentration inside the junctional SR falls to submillimolar levels during coupling of excitation to contraction.
authors.affiliation: Krannert Institute of Cardiology, Indianapolis, Indiana.
authors.forename: R D
authors.initials: RD
authors.lastname: Mitchell
authors.forename: H K
authors.initials: HK
authors.lastname: Simmerman
authors.forename: L R
authors.initials: LR
authors.lastname: Jones
mesh_terms: D000818:Animals; D001389:Azirines / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002155:Calsequestrin / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D009124:Muscle Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D001389:Azirines; D002155:Calsequestrin; D009124:Muscle Proteins; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D002118:Calcium