Oligomeric interactions between phospholamban molecules regulate Ca-ATPaseactivity in functionally reconstituted membranes

Phospholamban (PLB) is a major target of the beta- adrenergic cascade in th e heart, and functions as an endogenous inhibitor of Ca-ATPase transport ac tivity, To identify whether oligomeric interactions between PLB molecules a re involved in regulating Ca-ATPase transport activity, we have investigate d functional interactions between PLB and the Ca-ATPase in proteoliposomes of purified PLB functionally co-reconstituted with the SERCA2a isoform of t he Ca-ATPase isolated from cardiac sarcoplasmic reticulum (SR). The calcium sensitivity of this reconstituted preparation and functional stimulation b y cAMP-dependent protein kinase (PKA) are virtually identical to those of t he Ca-ATPase in cardiac SR microsomes, ensuring the functional relevance of this reconstituted preparation. Interactions between PLB molecules were me asured following covalent modification of the single lysine (i.e., Lys(3)) in PLB isolated from cardiac SR membranes with fluorescein isothiocyanate ( FITC) prior to co-reconstitution with the Ca-ATPase. FITC modification of P LB does not interfere with the ability of PLB to inhibit the Ca-ATPase, sin ce FITC-PLB co- reconstituted with the Ca-ATPase exhibits a similar calcium dependence of Ca-ATPase activation to that observed in native SR membranes . Thus, the functional arrangement of PLB with the Ca-ATPase is not modifie d by FITC modification. Using changes in the anisotropy of FITC-PLB resulti ng from fluorescence resonance energy transfer (FRET) between proximal PLB molecules to measure the average size and spatial arrangement of FITC chrom ophores, we find that PLB self-associates to form oligomers whose spatial a rrangement with respect to one another is in agreement with earlier suggest ions that PLB exists predominantly as a homopentamer. The inability of PKA to activate PLB following covalent modification with FITC permits functiona l interactions between PLB molecules associated with the Ca-ATPase activati on to be identified. A second-order loss of Ca-ATPase activation by PKA is observed as a function of the fractional contribution of FITC-PLB, indicati ng that PKA-dependent activation of two PLB molecules within a quaternary c omplex containing the Ca-ATPase is necessary for activation of the Ca-ATPas e. We suggest that the requirement for activation of two PLB molecules by P KA represents a physiological mechanism to ensure that activation of the Ca -ATPase following beta -adrenergic stimulation in the heart only occurs abo ve a threshold level of PKA activation.