FXYD protein isoforms differentially modulate human Na/K pump function

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.

FXYD proteins and sodium pump regulatory mechanisms

“…Crambert et al showed that PLM coimmunoprecipitated with the NKA in native heart and skeletal muscle and provided the first suggestion of PLM’s function. Electrophysiology ( Crambert et al, 2002 ; Meyer et al, 2020 ) and Na-sensitive dye ( Despa et al, 2005 ; Khafaga et al, 2012 ) measurements revealed PLM decreases the apparent affinity of NKA for intracellular sodium.…”

“…PLM phosphorylation by PKC at Ser69 (analogous to Thr69 residue in humans) has also been shown to increase PLM trafficking to the plasma membrane ( Lansbery et al, 2006 ), and we have recently presented evidence that PKA increases the plasma membrane density of human NKA heterologously expressed with human PLM in Xenopus laevis oocytes ( Meyer et al, 2020 ). In these scenarios, enhanced transport activity may be important when cardiac function increases during sympathetic stimulation.…”

“…Though there is a wide consensus that interaction with unphosphorylated PLM decreases the apparent Na + affinity of NKA and PLM phosphorylation increases the apparent Na + affinity of NKA ( Crambert et al, 2002 ; Bibert et al, 2008 ; Despa et al, 2005 ; Han et al, 2006 ; Meyer et al, 2020 ), the effect of PLM and PLM phosphorylation on V max are not fully established. Two studies showed that injection of synthesized unphosphorylated PLM cytoplasmic peptide (residues 54–72; Pavlović et al, 2007 ) or overexpression of human unphosphorylated PLM protein ( Mishra et al, 2015 ) decreases the V max of NKA.…”

“…Kuster et al (2000) used tryptic peptide mapping and mass spectrometry to show that the two distinct γ subunit variants, termed the γa subunit and γb subunit, differ in six residues at their N termini, with γa having two extra N-terminal residues. Both γ-subunit variants are primarily expressed in the thick ascending limb ( Arystarkhova et al, 2002b ) and induce identical reduction of the NKA apparent affinity for sodium ( Béguin et al, 2001 ; Arystarkhova et al, 1999 ; Therien et al, 1999 ; Béguin et al, 1997 ; Meyer et al, 2020 ). The γa and γb subunits do seem to have differential posttranslational modifications that have different effects on NKA kinetics ( Arystarkhova et al, 2002a ).…”

What FXYDs fix

“…However, the physiological effect of FXYD1 phosphorylation on acute stimulation of pump turnover in muscle remains elusive since FXYD1 knockout mice exhibit normal exercise capacity and no change in muscle contractility or fatigue (Manoharan et al, 2015). In the work reported earlier, Meyer et al (2020) found that coexpression with FXYD1 reduces the outward current produced, but that oocytes injected with PKA have similar currents regardless of coexpressed FXYD1. As they discussed, different groups have often reached opposing conclusions regarding the effects of FXYD1 phosphorylation, and it remains to be firmly established whether and how the Na,K ATPase ion affinity, maximal capacity, and membrane translocation are regulated by FXYD1 phosphorylation.…”