NA-SENSITIVE, OUABAIN-SENSITIVE, CA2+-SENSITIVE, AND THAPSIGARGIN-SENSITIVE ATPASE ACTIVITY EXPRESSED IN CHIMERAS BETWEEN THE CALCIUM AND THE SODIUM-PUMP ALPHA-SUBUNITS()

Using the chicken sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA)-ATPa se as a parental molecule and replacing various portions with the corr esponding portions of the chicken Na+,K+-ATPase alpha(1) subunit, Ca2/thapsigargin- and Na+/ouabain-sensitive domains critical for these P- type ATPase activities were identified. In the chimera, [n/c]CC, the a mino terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (S ERCA1) ATPase were replaced with the corresponding portion (Met-1-Asp- 200) of the Na+,K+-ATPase alpha(1) submit. In the chimera CC[c/n], the carboxyl-terminal amino acids (Ser-830 to COOH) of the SERCA1 ATPase were replaced with the corresponding segment (Leu-861 to COOH) of the Na+,K+-ATPase alpha(1) subunit, and in the chimera CNC, the middle par t (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na+,K-ATPase alpha(1) subunit (Gly-378-Lys-724). None of the chimeric molec ules exhibited any detectable ouabain-sensitive Na+,K+-ATPase activity , but they did exhibit thapsigargin-sensitive Ca2+-ATPase activity. Th erefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA 1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SER CA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was f urther stimulated by addition of Na+ in the assay medium containing Ca 2+. This additional stimulation of SERCA1-ATPase activity by Na+ was a bolished when the amino-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([Delta n/c]CC). In the absence of Na+, the SERCA1-ATPase acti vity of [n/c]CC was inhibited by ouabain, and, in the presence of Na+, its activity was stimulated by this drug. On the other hand, the ATPa se activity of [Delta n/c]CC was not affected by ouabain, although [De lta n/c]CC can still bind [H-3]ouabain. These results suggest that a d istinct Na+-sensitive domain (Na+ sensor) located within the restricte d amino-terminal region (Met-1-Leu-69) of the Na+,K+-ATPase alpha(1) s ubunit regulates ATPase activity. The Na+ sensor also controls ouabain action in concert with the major ouabain-binding region between Ala-7 0 and Asp-200 of alpha(1) subunit.