Date of Award

2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physiology

Abstract

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ into the endoplasmic reticulum of cells to establish a reservoir for Ca2+ signaling. In the heart, the activity of this transporter is tightly controlled via direct interactions with two competing regulatory micropeptides: phospholamban (PLB) and dwarf open reading frame (DWORF). PLB inhibits SERCA, while DWORF activates SERCA. These competing interactions determine cardiac performance by modulating the Ca2+ signals that drive the contraction/relaxation cycle. Previous studies indicated these SERCA-micropeptide interactions are Ca2+-sensitive; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. Here, FRET-microscopy demonstrated that this opposing Ca2+-sensitivity drives dynamic shifts in SERCA-micropeptide binding during cellular Ca2+ elevations. Evaluating the rates of these equilibrium shifts revealed that PLB monomers freed from SERCA during Ca2+ elevations rapidly oligomerize into PLB pentamers. These stable oligomers unbind slowly, delaying the rebinding of inhibitory PLB monomers to SERCA after Ca2+ elevations. In contrast, DWORF is exchanged rapidly on and off SERCA with respect to the rise and fall of transient Ca2+ signals. Computational modeling revealed that the slow unbinding of PLB pentamers causes PLB monomers to accumulate in these complexes during accelerated cardiac pacing. We propose that this accumulation of PLB pentamers decreases availability of inhibitory PLB monomers to bind SERCA and contributes to an increase in the contractile force of cardiac muscle at faster heart rates. Moreover, we demonstrated that a mutation of PLB, Arginine 14 deletion, which is associated with lethal dilated cardiomyopathy, further stabilizes PLB pentamers and blunts these dynamics adjustments to Ca2+ handling. It was also determined that the reciprocal Ca2+ sensitivity of PLB and DWORF results from their preference for binding different intermediate conformations that SERCA samples during Ca2+ transport. Specifically, PLB had the highest affinity for the ATP-bound state of SERCA, which prevails at low [Ca2+]. This result led us to hypothesize that tight binding of PLB to the ATP-bound state of SERCA may relate to its inhibitory effect on SERCA, decreasing the pump’s apparent Ca2+ affinity. Using a 2-color SERCA biosensor to report changes in SERCA conformation during Ca2+ binding by changes in intramolecular FRET, we tested whether PLB reduces SERCA Ca2+ affinity in the presence and absence of nucleotide. The results suggest that PLB inhibits SERCA through reversing an allosteric activation of the pump by ATP.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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