title: Carboxy-terminal regions of the sarcoplasmic/endoplasmic reticulum Ca(2+)- and the Na+/K(+)-ATPases control their K+ sensitivity.
abstract: The Na+,K(+)-ATPase and the sarcoplasmic/endoplasmic reticulum Ca(2+)-(SERCA-) ATPase belong to a family of P-type ATPases that undergo a cycle of conformational changes between the phosphorylated and dephosphorylated stages in an ion-specific manner. The ouabain-inhibitable Na+,K(+)-ATPase activity requires Na+ and K+. On the other hand, the Ca(2+)-dependent and thapsigargin-inhibitable activity of the SERCA-ATPase does not depend upon Na+ and K+ for its basal activity. However, the SERCA-ATPase and Ca(2+)-transport activities can be further activated either by K+ in a two-step fashion with high (ED50 approximately 20 mM) and low affinity (ED50 approximately 70 mM) or by Na+ in a one-step fashion with an ED50 value of approximately 50 mM. A chimera, in which the carboxy-terminal region (Leu861-COOH) of the Na+,K(+)-ATPase alpha 1 subunit replaced the corresponding region (Ser830-COOH) of the SERCA1-ATPase, lacked the low-affinity K+ activation of the SERCA-ATPase but displayed a higher-affinity (ED50 < 10 mM) activation by K+, similar to that of the Na+,K(+)-ATPase, whereas activation by Na+ was not affected. The replacement of the large cytosolic loop (Gly354-Lys712) and the amino-terminal regions (Met1-Asp162) of the SERCA1-ATPase with the corresponding portions of the Na+,K(+)-ATPase alpha 1 subunit did not affect the sensitivity of the SERCA-ATPase activity to K+. Thus, the carboxy-terminal regions of both the SERCA1 and the Na+,K(+)-ATPase alpha 1 subunit are critical for K+ sensitivity. Analysis of additional (Ca2+/Na+,K+)-ATPase chimeras demonstrated that the carboxy-terminal 102 amino acids (Phe920-Tyr1021) of the Na+/K(+)-ATPase alpha 1 subunit are sufficient to shift the K+ affinity for activation of the SERCA-ATPase without the beta subunit. No change in the two-step activation of SERCA-ATPase by K+ was seen when residues Thr871-Thr898 of the SERCA1-ATPase were replaced with residues Asn894-Ala919 of the Na+,K(+)-ATPase alpha 1 subunit, a region known to bind the Na+,K(+)-ATPase beta subunit [Lemas, M. V., et al. (1994) J. Biol. Chem. 269, 8255-8259]. Thus, the Na+,K(+)-ATPase subunit-assembly domain and the K(+)-sensitive region are distinct within the carboxy-terminal 161 amino acids of the Na+,K(+)-ATPase.
authors.affiliation: Department of Medical Biochemistry, Ohio State University, Columbus 43210, USA.
authors.forename: T
authors.initials: T
authors.lastname: Ishii
authors.forename: F
authors.initials: F
authors.lastname: Hata
authors.forename: M V
authors.initials: MV
authors.lastname: Lemas
authors.forename: D M
authors.initials: DM
authors.lastname: Fambrough
authors.forename: K
authors.initials: K
authors.lastname: Takeyasu
mesh_terms: D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D002846:Chromatography, Affinity; D007700:Kinetics; D007739:L Cells; D051379:Mice; D008954:Models, Biological; D008961:Models, Structural; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D010042:Ouabain; D019284:Thapsigargin; D012964:Sodium; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium