Entrance port for Na+ and K+ ions on Na+,K+-ATPase in the cytoplasmic loopbetween trans-membrane segments M6 and M7 of the alpha subunit - Proximityof the cytoplasmic segment of the beta subunit

Based on the following observations we propose that the cytoplasmic loop be tween trans-membrane segments M6 and M7 (L6/7) of the a subunit of Na+,K+-A TPase acts as an entrance port for Na+ and K+ ions. 1) In defined condition s chymotrypsin specifically cleaves L6/7 in the M5/M6 fragment of 19-kDa me mbranes, produced by extensive proteolysis of Na+,K+-ATPase, and in paralle l inactivates Rb+ occlusion, 2) Dissociation of the MB/MB fragment from 19- kDa membranes is prevented either by occluded cations or by competitive ant agonists 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 (Br-2-TITU3+). 3) Ca2+ io ns raise electrophoretic mobility of the M5/M6 fragment but not that of the other fragments of the alpha subunit, It appears that negatively charged r esidues in L6/7 recognize either Na+ or K+ ions or the competitive cation a ntagonists. Na+ and K+ ions are then occluded within trans-membrane segment s and can be transported, whereas the cation antagonists are not occluded a nd block transport at the entrance port. The cytoplasmic segment of the bet a subunit appears to be close to or contributes to the entrance port, as in ferred from the following observations. 1) Specific chymotryptic cleavage o f the 16-kDa fragment of the beta subunit to 15-kDa at 20 degrees C (Shains kaya, A., and Karlish, S, J, D, (1996) J, Biol, Chem. 271, 10309-10316) mar kedly reduces affinity for Br-2-TITU3+ and for Na+ ions, detected by Na+ oc clusion assays or electrogenic Na+ binding, whereas Rb+ occlusion is unchan ged. 2) Na+ ions specifically protect the 16-kDa fragment against this chym otryptic cleavage.