title: Entrance port for Na(+) and K(+) ions on Na(+),K(+)-ATPase in the cytoplasmic loop between trans-membrane segments M6 and M7 of the alpha subunit. Proximity Of the cytoplasmic segment of the beta subunit.
abstract: Based on the following observations we propose that the cytoplasmic loop between trans-membrane segments M6 and M7 (L6/7) of the alpha 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 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(2+), Mg(2+), La(3+), 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)-TITU(3+)). 3) Ca(2+) ions raise electrophoretic mobility of the M5/M6 fragment but not that of the other fragments of the alpha 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 beta 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 beta subunit to 15-kDa at 20 degrees C (Shainskaya, A., and Karlish, S. J. D. (1996) J. Biol. Chem. 271, 10309-10316) markedly reduces affinity for Br(2)-TITU(3+) 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.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel and Department of Biology, University of Konstanz, D-78434 Konstanz, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Shainskaya
authors.forename: A
authors.initials: A
authors.lastname: Schneeberger
authors.forename: H J
authors.initials: HJ
authors.lastname: Apell
authors.forename: S J
authors.initials: SJ
authors.lastname: Karlish
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002918:Chymotrypsin / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007550:Isothiuronium / Q000031:analogs & derivatives / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012413:Rubidium / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine; D013696:Temperature; D013997:Time Factors
chemical_list: C108416:1,3-dibromo-2,4,6-tris-(methylisothiouronium)benzene; D004791:Enzyme Inhibitors; D007550:Isothiuronium; D010042:Ouabain; D012964:Sodium; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011188:Potassium; D002118:Calcium