Novel approach for quantification of endoplasmic reticulum Ca²⁺transport

Abstract

The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+ 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 Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ 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. NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the cellular environment. The newly developed Ca2+ sensor R-CEPIA1er was used to successfully analyze Ca2+ 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 Ca2+ pump regulation.

Dwarf open reading frame (DWORF) peptide is a direct activator of the sarcoplasmic reticulum calcium pump SERCA

“…Moreover, it was noteworthy that DWORF enhanced the maximum ER calcium load ( Figure 4C, arrow). Since the thermodynamic driving force for calcium transport is the ATP/ADP ratio, we conclude that DWORF enhances SERCA catalytic efficiency (energetic cost of calcium transport) (28).…”

“…Stable cell lines were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum at 5% CO2 and 37 o C. Expression of SERCA2a in the stable cell line was verified by western blot analysis 48h after induction of recombinant pump expression with 1g/ml tetracycline. This strategy results in SERCA2a expressed 10-fold over endogenous SERCA2b (28), so calcium uptake is dominated by the exogenous cardiac isoform. MCer-DWORF expression in these cells showed a similar pattern as CEPIA-1er ( Figure 4A), indicating that DWORF is preferentially localized in the ER membrane.…”

“…The data suggested that DWORF not only relieves SERCA inhibition by displacing PLN, but it directly stimulates SERCA activity. To determine whether increased ATPase activity was accompanied by increased cellular calcium transport activity, we utilized a newly developed approach for measuring calcium uptake into the endoplasmic reticulum of live cells (28). The technique used an inducible human SERCA2a stable cell line (t-Rex-293 cells) with the calcium release channel RyR2 (allows manipulation of ER calcium load), and an ER-targeted calcium indicator R-CEPIA1er (allows measurement of ER calcium load) (28,29).…”

“…The cell line was transiently transfected with mCer-DWORF (or mCer-PLN) to assess regulation of SERCA2a. mCer-DWORF was predominantly expressed in the ER (estimated from the overlap between the mCer-DWORF and the R-CEPIA1er signals; Figure 4A), with a distribution pattern similar to SERCA (28). To determine SERCA2a function, the plasma membrane was selectively permeabilized with saponin to allow control of the cytosolic environment, including free calcium and ATP concentrations.…”

“…To determine SERCA2a function, the plasma membrane was selectively permeabilized with saponin to allow control of the cytosolic environment, including free calcium and ATP concentrations. ER calcium uptake was quantified from the increase in R-CEPIA1er fluorescence after full ER calcium depletion by caffeine followed by RyR2 inhibition with ruthenium red and tetracaine (RyR+Tetr; Figure 4B), which blocks the principal calcium leak pathway (28). The first derivative of ER calcium uptake (d[Ca 2+ ]ER/dt) was plotted against the corresponding [Ca 2+ ]ER to estimate the maximum ER calcium uptake rate and the maximum ER calcium load ( Figure 4C).…”