New Molecular Determinants Controlling the Accessibility of Ouabain to Its Binding Site in Human Na,K-ATPase Isoforms

Inhibition of Na,K-ATPase 2 isoforms in the human heart is supposed to be involved in the inotropic effect of cardiac glycosides, whereas inhibition of 1 isoforms may be responsible for their toxic effects. Human Na,K-ATPase 1 and 2 isoforms exhibit a high ouabain affinity but significantly differ in the ouabain association and dissociation rates. To identify the structural determinants that are involved in these differences, we have prepared chimeras between human 1 and 2 isoforms and 2 mutants in which nonconserved amino acids were exchanged with those of the 1 isoform, expressed these constructs in Xenopus laevis oocytes, and measured their ouabain binding kinetics. Our results show that replacement of Met and Ser in the M1–M2 extracellular loop of the 2 isoform by the corresponding Thr and Gln of the 1 isoform shifts both the fast ouabain association and dissociation rates of the 2 isoform to the slow ouabain binding kinetics of the 1 isoform. The amino acids at position 119 and 124 cooperate with the M7–M8 hairpin and are also responsible for the small differences in the ouabain affinity of the ouabainsensitive 1 and 2 isoforms. Thus, we have identified new structural determinants in the Na,K-ATPase -subunit that are involved in ouabain binding and probably control, in an isoform-specific way, the access and release of ouabain to and from its binding site. The ubiquitous Na,K-ATPase, which is responsible for the maintenance of the Na and K gradients in animal cells, functions as the pharmacological receptor for cardiac glycosides. Compounds such as digoxin, digitoxin, and ouabain are plant-derived steroids that bind to Na,K-ATPase with high selectivity and inhibit its transport activity. In myocardial cells, this inhibition results in a sequential increase in intracellular sodium and calcium concentrations and, consequently, an increase in the force of contraction. Especially digoxin is still widely used as an inotropic drug in the treatment of congestive heart failure despite its low therapeutic index. A better understanding of the structural features that determine cardiac glycoside interaction with Na,K-ATPase should help to develop inotropic drugs with better therapeutic effects and lower toxic effects. The Na,K-ATPase is composed of a catalytic -subunit with 10 transmembrane segments, which contains cation, ATP, phosphate, and cardiac glycoside binding sites as well as a -subunit that is a type II membrane protein required for the structural and functional maturation of the -subunit (Geering, 2001). Four and three isoforms exist that show a different tissue distribution (for review, see Blanco and Mercer, 1998) and that can produce Na,K-ATPase isozymes with different transport and pharmacological properties (Crambert et al., 2000). The contribution of different Na,K-ATPase isozymes to the pharmacological or toxic effects of cardiac glycosides is only partially understood. In rodents, the 1 isoform exhibits a nearly 1000-fold lower affinity for cardiac glycosides than 2 or 3 isoforms. Because rat cardiomyocytes express only 1 and 2 isoforms, it has been speculated that inhibition of the sensitive 2 isoform with low doses of cardiac glycoside produces the positive inotropic effect, whereas additional inhibition of 1 isoforms at higher doses leads to the toxic effect (Adams et al., 1982; Maixent et al., 1987). This hypothesis is supported by the recent observation that mouse hearts with genetically reduced levels of 2 isozymes are hypercontractile as a result of increased Ca transients, which mimics the inotropic effect of cardiac glycosides. On the other hand, mouse hearts with reduced levels of 1 isoforms are hypocontractile, which resembles cardiac glycoside toxicity (James et al., 1999). In humans, the situation is complicated by the fact that 1, 2, and 3 isoforms are present in the heart (Wang et al., 1996) and that all human isoforms have a similar high affinity for cardiac glycosides (Crambert et al., 2000; Wang et al., 2001). It has been speculated that an 2 isoform-specific function in the heart could be supported by a compartmentalization of the 2 isoform together with the Na /Ca exchanger into microdomains, near the sarco-/endoplasmic reticulum (Juhaszova and Blaustein, 1997). Another argument for a role of human 2 isoforms in the positive inotropic effect of cardiac glycosides is that ouabain binding to 1 This work was supported by Swiss National Science Foundation grant 31-64793.01 (to K.G.). 0026-895X/04/6502-335–341$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 2 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2923/1123086 Mol Pharmacol 65:335–341, 2004 Printed in U.S.A. 335 at A PE T Jornals on M ay 3, 2017 m oharm .aspeurnals.org D ow nladed from isoforms but not to 2 isoforms is efficiently antagonized by K at physiological concentrations (Crambert et al., 2000). Compared with 1 isoforms, human 2 isoforms display still another pharmacological difference that could be important for their implication in the inotropic effect of cardiac glycosides. Indeed, 2 isoforms have 5to 10-fold faster ouabain association and dissociation kinetics than 1 isoforms (Crambert et al., 2000). Because association and dissociation rates vary in parallel, the Kd values are similar for 1 and 2 isoforms. So far, only 1 isoforms have been studied with respect to the localization of the binding site and the molecular determinants of the association and dissociation kinetics of cardiac glycosides. Experimental and modeling data suggest that cardiac glycosides bind to the extracellular surface of the -subunit (Croyle et al., 1997; Middleton et al., 2000; Farr et al., 2002) and that the physical binding site for cardiac glycosides may be formed by the M3/M4 and M5/M6 hairpins (Koenderink et al., 2000). The association rate of ouabain seems to be dependent on the steroid moiety, whereas the dissociation rate depends both on the steroid and the sugar moieties (Yoda, 1974; Kawamura et al., 2001). For 1 isoforms, ouabain association rates are slow and independent of their sensitivity to cardiac glycosides, whereas dissociation rates are low in sensitive isoforms but high in resistant 1 isoforms (Yoda, 1974). In view of these data, it may be predicted that the differences in the ouabain association and dissociation rates in human 1 and 2 isoforms, which have a similar high sensitivity to cardiac glycosides, reflect differences in the accessibility of ouabain to its binding site rather than an alteration of the binding site itself. In this study, we aimed to characterize the structural determinants that influence association and dissociation rates of cardiac glycosides to Na,K-ATPase and in particular to the ‘inotropic’ 2 isoform. We produced chimeras between human 1 and 2 isoforms or replaced amino acids in the 2 isoform by the corresponding amino acids of the 1 isoform, expressed the mutants in Xenopus laevis oocytes, and determined the association and dissociation rate and the equilibrium binding constants for ouabain. Our results indicate that the M1–M2 and the M7–M8 hairpins contain several specific amino acids that determine the differences in the ouabain binding kinetics in 1 and 2 isoforms and control the access of ouabain to its binding site. Materials and Methods Mutants and Chimeras. Chimeras of human 1 and 2 isoforms were produced by introducing restriction sites into the 1 cDNA that are present in the 2 cDNA. polymerase chain reaction fragments of 1 cDNA were digested and ligated into the 2 cDNA to replace the corresponding regions (Table 1). Point mutations were introduced into the human Na,K-ATPase 2 isoform, previously cloned into the pSD5 vector (Crambert et al., 2000), by the polymerase chain reaction-based method described by Nelson and Long (1989). The nucleotide sequences of all constructs were confirmed by dideoxy sequencing and cRNAs were prepared by in vitro translation (Melton et al.,