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