The endoplasmic reticulum luminal Ca2+ regulates cardiac Ca2+ pump function

Abstract

The type 2a sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) plays a central role in Ca²⁺ signaling of cardiomyocytes. The speed at which SERCA2a pumps Ca²⁺ from the cytosol into the sarcoplasmic reticulum (SR) determines the diastolic relaxation rate. SERCA2a activity also sets SR Ca²⁺ load, which determines the amplitude of SR Ca²⁺ release and the systolic contraction strength. While SERCA2a controls the SR luminal [Ca²⁺] ([Ca²⁺]_SR), less is known about how dynamic changes in [Ca²⁺]_SR affect SERCA2a function. By measuring the endoplasmic reticulum [Ca²⁺] ([Ca²⁺]_ER) with the Ca²⁺ sensor R-CEPIA1er, we characterized the function of recombinant human and native mouse SERCA2a. We found that despite low endoplasmic reticulum (ER) Ca²⁺ gradient, SERCA2a-mediated Ca²⁺ transport was significantly slower at low [Ca²⁺]_ER than at intermediate [Ca²⁺]_ER. It appears that certain [Ca²⁺]_ER is required for optimal SERCA2a Ca²⁺ transport. We tested whether negatively charged amino acids within the luminal loop between transmembrane helices M7 and M8 contribute to SERCA2a regulation by [Ca²⁺]_ER. We found that the triple mutation E877L/D878L/E883L in the M7-M8 loop reduces SERCA2a Ca²⁺ transport particularly at intermediate [Ca²⁺]_ER. Destabilizing the M7-M8 loop by breaking a disulfide bond between cysteines 875 and 887 abolished ER Ca²⁺ transport. Complementary molecular dynamics simulations showed that the triple mutant E877L/D878L/E883L stabilizes a Ca²⁺-bound E2 state of the pump, slowing down release of Ca²⁺ from the transport sites into the ER compared with the wild-type SERCA2a. These results revealed, for the first time, that SERCA2a Ca²⁺ transport is regulated by the luminal Ca²⁺ by interacting with the M7-M8 loop.

Keywords: calcium ATPase; calcium pump; endoplasmic reticulum; fluorescent microscopy; intracellular calcium signaling.

Fig. 1.

SERCA2a-mediated Ca²⁺ uptake as a function of [Ca²⁺]_ER in HEK239 cells expressing human SERCA2a. A) Experimental protocol to measure SERCA-mediated ER Ca²⁺ uptake. A line-scan image of R-CEPIA1er (top) and corresponding profile of changes in [Ca²⁺]_ER (bottom) were measured in control conditions, during RyR2 activation with 5 mM caffeine with following RyR2 inhibition with 15 μM ruthenium red. In the end of each experiment, the R-CEPIA1er signal was converted to [Ca²⁺] after measuring F_max in the presence 5 mM Ca²⁺ and 5 μM ionomycin and F_min in the presence of 5 mM caffeine. Black line (Ctrl)—control conditions. Red line (TG)—thapsigargin. B) For each individual cell, ER Ca²⁺ uptake was analyzed as the first derivative (d[Ca²⁺]_ER/dt) and plotted as a function of ER Ca²⁺ load ([Ca²⁺]_ER). C) The average results of SERCA2a-mediated Ca²⁺ uptake rate at different ER Ca²⁺ loads: low Ca²⁺ loads were within the range 0.05–0.10 mM; intermediate (inter) loads were within 0.2–0.4 mM and high loads were within 0.6–0.8 mM. The analysis is based on results from 40 cells.

Fig. 2.

SERCA-mediated ER Ca²⁺ uptake during Ca²⁺ waves activated by relieving RyR2 inhibition. A) An example of Ca²⁺ waves recorded after relieving RyR2 inhibition by 10 mM cytosolic [Mg²⁺]. B) The average results of SERCA2a-mediated Ca²⁺ uptake rate during Ca²⁺ wave at different ER Ca²⁺ loads: low 0.05–0.10 mM; intermedium (inter) 0.2–0.4 mM and high 0.5–0.6 mM. The analysis is based on results from 20 cells.

Fig. 3.

SERCA2a-mediated Ca²⁺ uptake as a function of [Ca²⁺]_SR in mouse ventricular myocytes. A) Profile of changes in [Ca²⁺]_SR during RyR2 activation with 5 mM caffeine with following RyR2 inhibition with 15 μM ruthenium red. The R-CEPIA1er signal was converted to [Ca²⁺] after measuring F_max in the presence 5 mM Ca²⁺ and 5 μM ionomycin and F_min in the presence of 5 mM caffeine. B) For each individual cell, SR Ca²⁺ uptake was analyzed as the first derivative (d[Ca²⁺]_ER/dt) and plotted as a function of [Ca²⁺]_ER. C) The average results of SERCA2a-mediated Ca²⁺ uptake rate at different SR Ca²⁺ loads: low 0.05–0.10 mM; intermedium (inter) 0.2–0.4 mM and high 0.6–0.8 mM. The analysis is based on results from 11 myocytes.

Fig. 4.

SERCA2a-mediated Ca²⁺ uptake as a function of [Ca²⁺]_ER in HEK239 cells expressing SERCA2a^WT and three M7-M8 SERCA2a mutants. A) Left: analysis of the electrostatic potential SERCA2a map. Negative charges are labeled in red. Positive charges are labeled in blue. Right: position of two cysteines (green) and five NCA (red) in the M7–M8 loop. The list of three SERCA2a mutants with NCAs and single cysteine mutations. B) Changes in [Ca²⁺]_SR during RyR2 activation with following RyR2 inhibition in HE293 cells expressing SERCA2a^WT and different SERCA2a mutants: SERCA2a^C875A, SERCA2a^5NCA, and SERCA2a^3NCA. The R-CEPIA1er signal was converted to [Ca²⁺] after measuring F_max in the presence 5 mM Ca²⁺ and 5 μM ionomycin and F_min in the presence of 5 mM caffeine. C) ER Ca²⁺ uptake was analyzed as the first derivative (d[Ca²⁺]_ER/dt) and plotted as a function of [Ca²⁺]_ER. D) The average results of maximal ER Ca²⁺ uptake rate in HE293 cells expressing SERCA2a^WT and different SERCA2a mutants. The analysis is based on results from 40 cells for SERCA2a^WT, 9 cells for SERCA2a^5NCA and 24 cells for SERCA2a^3NCA.

Fig. 5.

Representation of translocation of a single Ca²⁺ ion from the transport sites of SERCA2a into the SR lumen. Structures of the SERCA2a embedded in a lipid bilayer extracted from the MD trajectories of the WT SERCA bound to a single Ca²⁺ ion. The head groups of the lipid bilayer are shown as white spheres; SERCA2a is shown as a gray cartoon representation. Key SERCA2a residues and the Ca²⁺ ion are shown as a van der Waals representation. We note that this translocation mechanism is inhibited upon mutation of residues E877, D878, and E883 to leucine.

Fig. 6.

Distance evolution between residues E90 and E309 with the Ca²⁺ ion occluded in the transport sites in the WT and triple mutant SERCA2a. Distances were calculated from each independent MD trajectory between the carbon delta (Cδ) of residues E90 (black trace) or E309 (red trace) and the Ca²⁺ ion. For these simulations, we used the crystal structure of SERCA2a representing the state at which Ca²⁺ is released into the SR lumen (PDB: 3b9b). This structure, which was obtained in the absence of Ca²⁺, a single Mg²⁺ ion was resolved in the transport sites; we replaced this ion for Ca²⁺ as a starting point for performing the atomistic MD simulations. The plots show that in SERCA2a^WT, Ca²⁺ partially (e.g. replicates 1 and 3) or fully (e.g. replicate 2) translocate into the luminal side of the lipid bilayer. Translocation is inhibited upon mutation of residues E877, D878, and E883 to leucine.