FXYD protein isoforms differentially modulate human Na/K pump function.

The Journal of general physiology (2020-11-25)
Dylan J Meyer, Sharan Bijlani, Marilina de Sautu, Kerri Spontarelli, Victoria C Young, Craig Gatto, Pablo Artigas
ABSTRACT

Tight regulation of the Na/K pump is essential for cellular function because this heteromeric protein builds and maintains the electrochemical gradients for Na+ and K+ that energize electrical signaling and secondary active transport. We studied the regulation of the ubiquitous human α1β1 pump isoform by five human FXYD proteins normally located in muscle, kidney, and neurons. The function of Na/K pump α1β1 expressed in Xenopus oocytes with or without FXYD isoforms was evaluated using two-electrode voltage clamp and patch clamp. Through evaluation of the partial reactions in the absence of K+ but presence of Na+ in the external milieu, we demonstrate that each FXYD subunit alters the equilibrium between E1P(3Na) and E2P, the phosphorylated conformations with Na+ occluded and free from Na+, respectively, thereby altering the apparent affinity for Na+. This modification of Na+ interaction shapes the small effects of FXYD proteins on the apparent affinity for external K+ at physiological Na+. FXYD6 distinctively accelerated both the Na+-deocclusion and the pump-turnover rates. All FXYD isoforms altered the apparent affinity for intracellular Na+ in patches, an effect that was observed only in the presence of intracellular K+. Therefore, FXYD proteins alter the selectivity of the pump for intracellular ions, an effect that could be due to the altered equilibrium between E1 and E2, the two major pump conformations, and/or to small changes in ion affinities that are exacerbated when both ions are present. Lastly, we observed a drastic reduction of Na/K pump surface expression when it was coexpressed with FXYD1 or FXYD6, with the former being relieved by injection of PKA's catalytic subunit into the oocyte. Our results indicate that a prominent effect of FXYD1 and FXYD6, and plausibly other FXYDs, is the regulation of Na/K pump trafficking.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Adenosine 5′-triphosphate magnesium salt, ≥95%, bacterial
Sigma-Aldrich
Collagenase from Clostridium histolyticum, lyophilized powder, ≥125 CDU/mg solid (CDU = collagen digestion units), 0.5-5.0 FALGPA units/mg solid
Sigma-Aldrich
Protein Kinase A Catalytic Subunit from bovine heart, ≥9 units/μg protein (cyclic-AMP is not required for this activity), lyophilized (white powder to sticky mass to hard pellet)
Sigma-Aldrich
Protein Kinase A Inhibitor Fragment 6-22 amide, ≥97% (HPLC)
Sigma-Aldrich
Anti-FXYD1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution