Novel approach for quantification of endoplasmic reticulum Ca transport.

Authors:
Elisa Bovo
Elisa Bovo
Loyola University Chicago
United States
Daniel Kahn
Daniel Kahn
Duke University Medical Center
United States
Quan Cao
Quan Cao
Qingdao Institute of Bioenergy and Bioprocess Technology
China
Jody L Martin
Jody L Martin
Loyola University Chicago
United States
Ivana Y Kuo
Ivana Y Kuo
John Curtin School of Medical Research
Australia
Seth L Robia
Seth L Robia
Loyola University Chicago
United States

Am J Physiol Heart Circ Physiol 2019 Jun 22;316(6):H1323-H1331. Epub 2019 Mar 22.

Department of Cell and Molecular Physiology, Loyola University Chicago , Chicago, Illinois.

The type 2a sarco-/endoplasmic reticulum Ca-ATPase (SERCA2a) plays a key role in Ca regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca sensor R-CEPIA1er was used to measure the concentration of Ca in the lumen of the endoplasmic reticulum (ER) ([Ca]) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca release channel ryanodine receptor (RyR2) created a Ca release/reuptake system that mimicked aspects of cardiac myocyte Ca handling. SERCA2a function was quantified from the rate of [Ca] refilling after ER Ca depletion; then, ER Ca leak was measured after SERCA inhibition. ER Ca uptake and leak were analyzed as a function of [Ca] to determine maximum ER Ca uptake rate and maximum ER Ca load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca-ATPase (SERCA) function in the cellular environment. The newly developed Ca sensor R-CEPIA1er was used to successfully analyze Ca uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca pump regulation.

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Source
https://www.physiology.org/doi/10.1152/ajpheart.00031.2019
Publisher Site
http://dx.doi.org/10.1152/ajpheart.00031.2019DOI Listing
June 2019
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