Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

Abstract

The Na,K-ATPase α subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca, whereas α has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α is preferentially expressed with β in T-tubules of cardiac myocytes, forming αβ heterodimers. We have expressed human αβ, αβ, αβ, and αβ in Pichia pastoris, purified the complexes, and compared their functional properties. αβ and αβ differ significantly from both αβ and αβ in having a higher KK and lower KNa for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K and shift of the EP-EP conformational equilibrium toward EP. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of αβ and αβ over αβ (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K affinity of αβ could allow an acute response to raised ambient K concentrations in physiological conditions and explain the importance of αβ for cardiac muscle contractility. The high sensitivity of αβ to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of αβ-selective digoxin derivatives for reducing cardiotoxicity.

An Efficient One‐Pot Enzymatic Synthesis of Cardiac Glycosides with Varied Sugar Chain Lengths

“…NKA includes four isoforms of the α unit (α1, α2, α3, and α4) and three isoforms of the β unit (β1, β2, and β3) as well as regulatory FXYD proteins . The α2 isoform is thought to play an important role in cardiac function and could be an attractive target for an isoform‐selective CTS . It has recently been hypothesized that α2 isoform‐selective CTS could induce cardiac contraction with minimal systemic toxicity and could be used as safer cardiotonic agents compared to conventional CTS .…”

The Structure and Function of the Na,K-ATPase Isoforms in Health and Disease

“…In addition to the four alpha isoforms, mammals express three beta and seven FXYD subunit isoforms. The combination of alpha1 and beta1 is expressed most widely (table), and any alpha can be expressed with any beta to yield a functional pump in Xenopus oocytes (Crambert et al, 2000 ; Hilbers et al, 2016 ), though in vivo the associations may be more selective (Tokhtaeva et al, 2012 ; Habeck et al, 2016 ).…”

How does pressure overload cause cardiac hypertrophy and dysfunction? High-ouabain affinity cardiac Na+pumps are crucial

“…Notably, ␣ 1 is widely distributed in the plasma membrane (PM) and is the "housekeeper" that maintains the low global cytosolic Na ϩ concentration ([Na ϩ ] CYT ) (6,62). The ␣ 1 -to-␣ 2 ratio (Ϸ5-20:1) is similar in rodents and humans, but rodent ␣ 1 -Na ϩ pumps are unusually resistant (R) to ouabain (8,32,54,70), i.e., the wild-type (WT) mouse genotype is ␣ 1 R/R ␣ 2 S/S (where S indicates ouabain sensitive).…”