Functional Properties of Na,K-ATPase, and Their Structural Implications, as Detected with Biophysical Techniques

A full understanding of the molecular mechanism of ion transport and energetics of the Na,K-ATPase will require both structural and functional data. During recent years biophysical methods have provided a number of important pieces of information on ion binding and release and the charge transfer process. This allows the formulation of kinetic models of the transport process. Although a breakthrough has not been obtained due to the lack of detailed knowledge on the threedimensional structure with a resolution high enough to identify the ion-binding sites and the transport pathway, the functional information has structural implications that create constraints on possible mechanisms of active transport. Here we describe briefly contributions of some biophysical methods to our conceptual understanding of the ion transport process

Binding of Sodium Ions and Cardiotonic Steroids to Native and Selectively Trypsinized Na,K Pump, Detected by Charge Movements

A fluorescent dye, RH421, has been used to characterize charge movements associated with cation and cardiotonic steroid binding to Na,K-ATPase and to a specifically trypsinized preparation, so-called "19-kDa membranes." A fluorescence decrease induced by Na+ is attributed to electrogenic binding of one Na+ ion from the cytoplasm. The apparent affinity for Na+ is the same in both preparations. (ATP + Na + Mg) or (Pi + Mg)-induced fluorescence signals observed with native enzyme are not observed in 19-kDa membranes, consistent with loss of ATP binding and phosphorylation. Cardiotonic steroids (CS) bind to native enzyme and 19-kDa membranes as judged by RH421 signals, fluorescence of anthroyl ouabain, anidn hibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and bindingis prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissociation are identical in both preparations, but aglycones bind and dissociate about 6-foldf aster to 19-kDa membranes. Binding of Na+ and cardiotonic steroids is inactivated upon heating or extensive Pronase digestion of 19-kDa membranes. This suggests that cation and CS binding depend on the structural integrity of a complex of the proteolytic fragments, and that sites for both cations or CS consist of ligating groups located on more than one fragments of 19-kDa membranes

Binding of sodium ions and cardiotonic steroids to native and selectively trypsinized Na,K pump, detected by charge movements

A fluorescent dye, RH421, has been used to characterize charge movements associated with cation and cardiotonic steroid binding to Na,K-ATPase and to a specifically trypsinized preparation, so-called "19-kDa membranes." A fluorescence decrease induced by Na+ is attributed to electrogenic binding of one Na+ ion from the cytoplasm. The apparent affinity for Na+ is the same in both preparations. (ATP + Na + Mg) or (Pi + Mg)-induced fluorescence signals observed with native enzyme are not observed in 19-kDa membranes, consistent with loss of ATP binding and phosphorylation. Cardiotonic steroids (CS) bind to native enzyme and 19-kDa membranes as judged by RH421 signals, fluorescence of anthroyl ouabain, anidn hibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and bindingis prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissociation are identical in both preparations, but aglycones bind and dissociate about 6-foldf aster to 19-kDa membranes. Binding of Na+ and cardiotonic steroids is inactivated upon heating or extensive Pronase digestion of 19-kDa membranes. This suggests that cation and CS binding depend on the structural integrity of a complex of the proteolytic fragments, and that sites for both cations or CS consist of ligating groups located on more than one fragments of 19-kDa membranes

Entrance Port for Na+ and K+ Ions on Na+,K+-ATPase in the Cytoplasmic loop between trans-membrane segments M6 and M7 of the α subunit : proximity of the cytoplasmic segment of the β subunit

Based on the following observations we propose that the cytoplasmic loop between trans-membrane segments M6 and M7 (L6/7) of the α subunit of Na+,K+-ATPase acts as an entrance port for Na+ and K+ ions. 1) In defined conditions chymotrypsin specifically cleaves L6/7 in the M5/M6 fragment of 19-kDa membranes, produced by extensive proteolysis of Na+,K+-ATPase, and in parallel inactivates Rb1 occlusion. 2) Dissociation of the M5/M6 fragment from 19-kDa membranes is prevented either by occluded cations or by competitive antagonists such as Ca²+, Mg²+, La³+, p-xylylene bisguanidinium and m-xylylene bisguanidinium, or 1-bromo- 2,4,6-tris(methylisothiouronium)benzene and 1,3-dibromo-2,4,6-tris (methylisothiouronium)benzene (Br2-TITU³1). 3) Ca²1 ions raise electrophoretic mobility of the M5/M6 fragment but not that of the other fragments of the a subunit. It appears that negatively charged residues in L6/7 recognize either Na+ or K+ ions or the competitive cation antagonists. Na+ and K+ ions are then occluded within trans-membrane segments and can be transported, whereas the cation antagonists are not occluded and block transport at the entrance port. The cytoplasmic segment of the β subunit appears to be close to or contributes to the entrance port, as inferred from the following observations. 1) Specific chymotryptic cleavage of the 16-kDa fragment of the β subunit to 15-kDa at 20°C (Shainskaya, A., and Karlish, S. J. D. (1996) J. Biol. Chem. 271, 10309 10316) markedly reduces affinity for Br2-TITU³+ and for Na+ ions, detected by Na+ occlusion assays or electrogenic Na+ binding, whereas Rb+ occlusion is unchanged. 2) Na+ ions specifically protect the 16-kDa fragment against this chymotryptic cleavage