Cardiac glycosides and isoforms of Na/K-ATPase 1 Selectivity of digitalis glycosides for isoforms of human Na,K-ATPase

There are four isoforms of the α subunit (α1-4) and three isoforms of the β subunit (β1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. α2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An α2selective cardiac glycoside could provide important insights into physiological and pharmacological properties of α2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing α1β1, α2β1 and α3β1 isoforms of human Na,K-ATPase expressed in P. pastoris and the purified detergentsoluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, β-methyl digoxin and digitoxin show moderate but highly significant selectivity (up to 4-fold) for α2/α3 over α1 (KDα1>α2=α3). By contrast, ouabain shows moderate selectivity (≈2.5-fold) for α1 over α2 (KDα1≤α3<α2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested, are indistinguishable (KDα1=α3=α2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure http://www.jbc.org/cgi/doi/10.1074/jbc.M110.119248 The latest version is at JBC Papers in Press. Published on April 13, 2010 as Manuscript M110.119248 Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on A uust 7, 2017 hp://w w w .jb.org/ D ow nladed from Cardiac glycosides and isoforms of Na/K-ATPase 2 of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately α2-selective. This supports a major role of α2 in cardiac contraction and cardiotonic effects of digitalis glycosides. Introduction For over two hundred years congestive heart failure has been treated with the plantderived digitalis cardiac glycosides such as digoxin, which increase force of contraction of failing cardiac muscle and reduce cardiac conduction rate. However, digoxin is now less used than in the past, due to the narrow therapeutic window and drug toxicity. Cardiac glycosides are produced in mammals as well as plants. As reviewed recently (1) five different cardiac glycosides have been identified in mammalian tissues including the cardenolides ouabain and digoxin and the bufodienolides marinobufagenin, telocinobufagin and 19norbufalin. Cardiac glycosides inhibit Na,KATPase and control cellular Na and K gradients, and also activate cellular signaling pathways that mediate control of gene expression and tissue growth (2,3). Thus a multiplicity of physiological functions could be affected by the multiplicity of endogenous cardiac glycosides but this is not well understood (1,4,5). The Na,K-ATPase consists of both α and β subunits, including four isoforms of α (α1, α2, α3, α4) and three isoforms of β (β1, β2, β3 ) (6), and FXYD (1-7) proteins, which are accessory subunits (7). α1 is almost ubiquitously distributed, while other isoforms are expressed in a tissueand development-specific fashion. α2 is found mainly in muscle (skeletal, smooth and cardiac), α3 primarily in nervous tissue and α4 only in testicles. Human heart expresses α1, α2 and α3 isoforms and β1 (8). In cardiac myocytes, α2 is concentrated in the T-tubules adjacent to the sarcoplasmic reticulum (SR), while α1 is more evenly distributed in T-tubules and SR. (9). In smooth muscle and brain astrocytes, α1 is uniformly distributed but α2 and α3 show a punctuated distribution, overlying the SR (10). Increased force of contraction of cardiac muscle induced by cardiac glycosides is the result of inhibition of Na,K-ATPase. Raised intracellular Na concentration limits Ca extrusion via the 3Na/Ca exchanger, leading to enhanced Ca uptake into the sarcoplasmic reticulum (SR) by the CaATPase, and increased Ca-induced Carelease during excitation-contraction coupling. Cardiac glycosides-induced toxicity is associated with excessive inhibition of Na,K-ATPase, accumulation of Ca ions (i.e. ”Ca-overload”) and “spontaneous” SR Ca release that can trigger delayed after-depolarizations and cardiac arrhythmias (11). Experiments with mice engineered to have ouabain-sensitive α1 and insensitive α2 isoforms (α1α2) or ouabain-insensitive α1 and α2 (α1α2), instead of the wild type with ouabain-insensitive α1 and ouabainsensitive α2 (α1α2), have shown that α2 can play a predominant role in cardiac glycosides-induced positive inotropy (12), and also that α2 carries a large fraction of the Na,K-pump current in T-tubules of cardiac myocytes (13). Very recent work shows that α2 preferentially modulates Ca transients and SR Ca release in cardiac myocytes compared to α1 (14). Similarly, α2 plays an important role in contractility of vascular smooth muscle and control of blood pressure (15,16). Experiments with α1α2, α1α2 and α1α2 mice also provide strong evidence for endogenous 1 Abbreviations: SR, sarcoplasmic reticulum; DDM, n-dodecyl-β-D-maltopyranoside; C12E8, octaethylene glycerol mondodecyl ether; SOPS (1-Stearoyl-2Oleoyl-sn-Glycero-3-[Phosphor-L-Serine]; CG, cardiac glycosides; PMSF, phenylmethylsulfonyl fluoride ; BCA, Bicinchoninic Acid ; DMSO, Dimethyl sulfoxide ; BP, blood pressure; by gest on A uust 7, 2017 hp://w w w .jb.org/ D ow nladed from Cardiac glycosides and isoforms of Na/K-ATPase 3 mammalian cardiac glycosides, by showing differential responses to physiological stimuli and states that alter blood pressure (12,17). In view of the accumulated information on α1 and α2 isoforms, one could envisage that α2-selective cardiac glycosides could provide important insights into the physiological role of α2 and, in particular, complement the information obtained from genetically engineered mice. At the pharmacological level an α2-selective cardiac glycoside could induce cardiac contraction with minimal Ca-overload, and so act as a safer cardiotonic agent than conventional cardiac glycosides. Structure-activity analyses of inhibition of Na,K-ATPase by cardiac glycosides or displacement of bound H-ouabain have been conducted for many years, see for example (18-22). As summarized in (23) essential structural features of active cardiac glycosides include: rings A/B and C/D are cis fused and B/C rings are trans fused, a hydroxyl group at C14, and an unsaturated lactone attached at C17 of the steroid. The sugar at C3 of the steroid is not essential for inhibition, but strongly affects binding affinity and rates, see Table 1B and (23). Despite the great potential interest in isoform selectivity of cardiac glycosides there is little clear-cut information. Variations in potency of digitoxigenin monoglycosides as inhibitors of kidney (α1β1) and brain (α1, α2, α3) Na,KATPases were attributed to isoform selectivity and a role of the sugar was proposed (24). However, most native tissues, except kidney (α1β1), contain mixtures of isoforms, and it is difficult to differentiate interactions of cardiac glycosides with the individual isoforms, and exclude possible complicating factors. Since, for example, human cardiac membranes express α1, α2 and α3 isoforms (8), healthy cardiac tissue would not be useful for this purpose even if it was readily available. Individual human isoforms have been expressed in Xenopus oocytes (α1-3, β1-3) (25) and S. cerevisae (α1-3, β1) (26,27) at low levels and used to characterize H-ouabain binding. More recently we have expressed human isoforms (α1,α2, and now also α3, with β1) at high levels in P. pastoris and purified the α1β1, α2β1 and α3β1 protein complexes (28-31). This system permits accurate analysis of cardiac glycoside binding and inhibition of Na,K-ATPase activity of the individual human isoforms, leading to the finding that several digitalis glycosides show moderate but highly significant selectivity for α2 (and