Entrance Port for Na and K Ions on Na,K-ATPase in the Cytoplasmic Loop between Trans-membrane Segments M6 and M7 of the a Subunit PROXIMITY OF THE CYTOPLASMIC SEGMENT OF THE b SUBUNIT*

Based on the following observations we propose that the cytoplasmic loop between trans-membrane segments M6 and M7 (L6/7) of the a subunit of Na,KATPase 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 Rb occlusion. 2) Dissociation of the M5/M6 fragment from 19-kDa membranes is prevented either by occluded cations or by competitive antagonists such as Ca, Mg, La, p-xylylene bisguanidinium and m-xylylene bisguanidinium, or 1-bromo2,4,6-tris(methylisothiouronium)benzene and 1,3-dibromo-2,4,6-tris (methylisothiouronium)benzene (Br2TITU). 3) Ca 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 b 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 b 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-TITU 31 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.