title: Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking.
abstract: Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys(27) of PLB was cross-linked to the Ca(2+) pump at the cytoplasmic extension of M4 (at or near Lys(328)) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca(2+) or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca(2+) affinity conformation of the Ca(2+)-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca(2+)-ATPase activity and Ca(2+) transport. The results suggest that the inhibitory effect of PLB on Ca(2+)-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca(2+) to the Ca(2+) pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca(2+) pump are largely unchanged in failing myocardium.
authors.affiliation: Krannert Institute of Cardiology and the Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Brandy L
authors.initials: BL
authors.lastname: Akin
authors.forename: Larry R
authors.initials: LR
authors.lastname: Jones
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport; D008861:Microsomes / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism* / Q000473:pathology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D018411:Spodoptera; D019284:Thapsigargin / Q000378:metabolism; D000099211:Phospholamban
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D002118:Calcium; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D009124:Muscle Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin; D000099211:Phospholamban; C506946:ATP2A2 protein, human