Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells

Area of Science:

• Biochemistry
• Molecular Biology
• Cardiovascular Physiology

Background:

• The sodium-potassium ATPase (Na/K-ATPase, NKA) is vital for cellular ion gradients, supporting essential physiological processes.
• NKA function involves alpha and beta subunits, modulated by FXYD proteins like phospholemman (PLM) in the heart.
• The precise stoichiometry of the NKA-PLM regulatory complex in live cells remains undetermined.

Purpose of the Study:

• To elucidate the structure and stoichiometry of the NKA-PLM regulatory complex in intact live cells.
• To investigate the binding affinities between NKA subunits and PLM.
• To develop a structural model of the NKA-PLM complex.

Main Methods:

• Fluorescence lifetime imaging and spectroscopy were employed to study the NKA-PLM complex.
• Concentration-dependent binding assays were performed.
• Docking and molecular dynamics (MD) simulations were utilized for structural modeling.

Main Results:

• Observed concentration-dependent subunit interactions within the NKA-PLM complex.
• Demonstrated avid association between alpha and beta subunits, with lower affinity alpha-alpha and alpha-PLM interactions.
• Established the stoichiometry of the regulatory complex as two alpha, two beta, and two PLM subunits (α₂β₂PLM₂).

Conclusions:

• The NKA-PLM regulatory complex in live cells exhibits a precise (α₂β₂PLM₂) stoichiometry.
• Alpha-alpha subunit interactions may facilitate conformational coupling, potentially enhancing NKA turnover.
• Impaired NKA expression in heart failure might hinder the formation of this complete regulatory complex, impacting cardiac function.