Figure 2.

Dynamics of PLB and DWORF binding to SERCA during elevations in intracellular Ca²⁺.A, a simplified Post-Albers scheme of the SERCA enzymatic cycle, highlighting states that predominate at low (blue) and high (red) intracellular [Ca²⁺]. B, FRET-based binding curves displaying a shifted dissociation constant (K_D) of PLB–SERCA binding between the ATP-bound (blue) and TG-bound (black) states of SERCA. C and D, apparent K_Ds of PLB or DWORF binding to different SERCA enzymatic states of the catalytic cycle as in panel (A) with lines representing mean ± SEM (n = 6). Ligands used to stabilize each state are shown in parentheses. Differences in micropeptide K_Ds between SERCA states were analyzed by one-way ANOVA with Tukey’s post hoc (∗p < 0.05). E, apparent K_Ds of PLB and DWORF for SERCA in ATP-containing solutions with low (blue) and high (red) concentrations of intracellular Ca²⁺ with lines representing mean ± SEM (n = 5). Differences in K_D evaluated by Student’s t test. F, confocal microscopy quantification of intracellular Ca²⁺ measured by X-rhod-1 fluorescence (gray raw data, with black smoothed trendline) with simultaneous measurement of changes in PLB–SERCA FRET (YFP/Cer ratio) (gray raw data, with blue smoothed trendline). G, quantification of ER luminal Ca²⁺ measured by R-CEPIA1er fluorescence with simultaneous measurement of PLB–SERCA FRET (YFP/Cer ratio). H, quantification of intracellular Ca²⁺ measured by X-rhod-1 fluorescence with simultaneous measurement of DWORF–SERCA FRET (YFP/Cer ratio) I, quantification of ER luminal Ca²⁺ measured by R-CEPIA1er fluorescence with simultaneous measurement of DWORF–SERCA FRET (YFP/Cer ratio). DWORF, dwarf ORF; ER, endoplasmic reticulum; PLB, phospholamban; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase.