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)-ATPase as a parental molecule and replacing various portions with the corresponding 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 amino terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (SERCA1) 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 part (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 molecules exhibited any detectable ouabain-sensitive Na+,K+-ATPase activity, but they did exhibit thapsigargin-sensitive Ca2+-ATPase activity. Therefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SERCA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was further stimulated by addition of Na+ in the assay medium containing Ca2+. This additional stimulation of SERCA1-ATPase activity by Na+ was abolished 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 activity 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 ATPase activity of [Delta n/c]CC was not affected by ouabain, although [Delta n/c]CC can still bind [H-3]ouabain. These results suggest that a distinct Na+-sensitive domain (Na+ sensor) located within the restricted amino-terminal region (Met-1-Leu-69) of the Na+,K+-ATPase alpha(1) subunit regulates ATPase activity. The Na+ sensor also controls ouabain action in concert with the major ouabain-binding region between Ala-70 and Asp-200 of alpha(1) subunit.