Investigating the Quaternary Structure of the SERCA-Micropeptide Regulatory Complex AHA Conference Repository

American Heart Association

Final ID: Wed133

Investigating the Quaternary Structure of the SERCA-Micropeptide Regulatory Complex

Abstract Body:

The sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA) is responsible for pumping cytosolic calcium into the sarcoplasmic reticulum (SR), enabling heart relaxation and establishing SR calcium load for heart contraction . SERCA2a activity decreases in heart failure (HF), making the protein and its regulatory network a prominent therapeutic target. To assess the interaction of SERCA with its micropeptide inhibitor phospholamban (PLB) and activator dwarf open reading frame (DWORF), we transfected HEK293 cells with OFP-SERCA and either mMaroon-PLB or mMaroon-DWORF and quantified Förster resonance energy transfer (FRET using time-correlated single photon counting (TCSPC). We generated fluorescence lifetime imaging microscopy (FLIM) images and performed extended analysis of single cells. Results for both micropeptides show multiple populations of donors in the multi-exponential fitting of the TCSPC decay. Overall, the data suggest two micropeptide acceptors bound to a dimeric complex of two donor-labeled SERCA pumps. This is consistent with a model in which SERCA forms conformationally coupled homodimers to enhance SERCA transport function. For PLB, we suggest a preliminary model in which one PLB initially binds to a high affinity site on one SERCA in the homodimer, generating distances of 57 and 82 Å from each SERCA donor to the PLB acceptor. At higher protein concentrations, a second PLB binds to a low affinity site on the second SERCA in the homodimer, quenching its interaction with the first PLB that bound and generating a 46Å distance that replaces the 82Å distance. For DWORF, our results are consistent with a model in which two DWORFs bind concurrently to the SERCA homodimer with similar affinity, generating distances of 49 and 67Å. Ongoing work is incorporating other micropeptides into these experiments, as well as SERCA-micropeptide regulatory complex changes on a beat-to-beat basis in neonatal rat ventricular myocytes.

Kersemeier, Hannah ( Loyola University Chicago , Oak Park , Illinois , United States )
Mazzenga, Alexis ( Loyola University Chicago , Oak Park , Illinois , United States )
Seflova, Jaroslava ( Loyola University Chicago , Maywood , Illinois , United States )
Robia, Seth ( Loyola University Chicago , Oak Park , Illinois , United States )

Author Disclosures:

Meeting Info:

Basic Cardiovascular Sciences 2026

2026

Boston, Massachusetts

Session Info:

Poster Session 3

Wednesday, 07/15/2026 , 04:30PM - 07:00PM

Poster Session and Reception

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