Oligomeric Interactions between Phospholamban Molecules Regulate Ca-ATPase Activity in Functionally Reconstituted Membranes
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- 作者：Qing Yao ; Linda T. L. Chen ; Jinhui Li ; Kimberley Brungardt ; Thomas C. Squier ; and Diana J. Bigelow
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：May 29, 2001
- 年：2001
- 卷：40
- 期：21
- 页码：6406 - 6413
- 全文大小：87K
- 年卷期：v.40,no.21(May 29, 2001)
- ISSN：1520-4995
文摘
Phospholamban (PLB) is a major target of the 
-adrenergic cascade in the heart, and functionsas an endogenous inhibitor of Ca-ATPase transport activity. To identify whether oligomeric interactionsbetween PLB molecules are involved in regulating Ca-ATPase transport activity, we have investigatedfunctional interactions between PLB and the Ca-ATPase in proteoliposomes of purified PLB functionallyco-reconstituted with the SERCA2a isoform of the Ca-ATPase isolated from cardiac sarcoplasmic reticulum(SR). The calcium sensitivity of this reconstituted preparation and functional stimulation by cAMP-dependent protein kinase (PKA) are virtually identical to those of the Ca-ATPase in cardiac SR microsomes,ensuring the functional relevance of this reconstituted preparation. Interactions between PLB moleculeswere measured following covalent modification of the single lysine (i.e., Lys3) in PLB isolated fromcardiac SR membranes with fluorescein isothiocyanate (FITC) prior to co-reconstitution with theCa-ATPase. FITC modification of PLB does not interfere with the ability of PLB to inhibit theCa-ATPase, since FITC-PLB co-reconstituted with the Ca-ATPase exhibits a similar calcium dependenceof Ca-ATPase activation to that observed in native SR membranes. Thus, the functional arrangement ofPLB with the Ca-ATPase is not modified by FITC modification. Using changes in the anisotropy ofFITC-PLB resulting from fluorescence resonance energy transfer (FRET) between proximal PLB moleculesto measure the average size and spatial arrangement of FITC chromophores, we find that PLB self-associates to form oligomers whose spatial arrangement with respect to one another is in agreement withearlier suggestions that PLB exists predominantly as a homopentamer. The inability of PKA to activatePLB following covalent modification with FITC permits functional interactions between PLB moleculesassociated with the Ca-ATPase activation to be identified. A second-order loss of Ca-ATPase activationby PKA is observed as a function of the fractional contribution of FITC-PLB, indicating that PKA-dependent activation of two PLB molecules within a quaternary complex containing the Ca-ATPase isnecessary for activation of the Ca-ATPase. We suggest that the requirement for activation of two PLBmolecules by PKA represents a physiological mechanism to ensure that activation of the Ca-ATPasefollowing -adrenergic stimulation in the heart only occurs above a threshold level of PKA activation.
-adrenergic cascade in the heart, and functionsas an endogenous inhibitor of Ca-ATPase transport activity. To identify whether oligomeric interactionsbetween PLB molecules are involved in regulating Ca-ATPase transport activity, we have investigatedfunctional interactions between PLB and the Ca-ATPase in proteoliposomes of purified PLB functionallyco-reconstituted with the SERCA2a isoform of the Ca-ATPase isolated from cardiac sarcoplasmic reticulum(SR). The calcium sensitivity of this reconstituted preparation and functional stimulation by cAMP-dependent protein kinase (PKA) are virtually identical to those of the Ca-ATPase in cardiac SR microsomes,ensuring the functional relevance of this reconstituted preparation. Interactions between PLB moleculeswere measured following covalent modification of the single lysine (i.e., Lys3) in PLB isolated fromcardiac SR membranes with fluorescein isothiocyanate (FITC) prior to co-reconstitution with theCa-ATPase. FITC modification of PLB does not interfere with the ability of PLB to inhibit theCa-ATPase, since FITC-PLB co-reconstituted with the Ca-ATPase exhibits a similar calcium dependenceof Ca-ATPase activation to that observed in native SR membranes. Thus, the functional arrangement ofPLB with the Ca-ATPase is not modified by FITC modification. Using changes in the anisotropy ofFITC-PLB resulting from fluorescence resonance energy transfer (FRET) between proximal PLB moleculesto measure the average size and spatial arrangement of FITC chromophores, we find that PLB self-associates to form oligomers whose spatial arrangement with respect to one another is in agreement withearlier suggestions that PLB exists predominantly as a homopentamer. The inability of PKA to activatePLB following covalent modification with FITC permits functional interactions between PLB moleculesassociated with the Ca-ATPase activation to be identified. A second-order loss of Ca-ATPase activationby PKA is observed as a function of the fractional contribution of FITC-PLB, indicating that PKA-dependent activation of two PLB molecules within a quaternary complex containing the Ca-ATPase isnecessary for activation of the Ca-ATPase. We suggest that the requirement for activation of two PLBmolecules by PKA represents a physiological mechanism to ensure that activation of the Ca-ATPasefollowing -adrenergic stimulation in the heart only occurs above a threshold level of PKA activation.