Na⁺-, ouabain-, Ca²⁺-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump α 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 α₁ 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 α₁ subunit. 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 at subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na⁺,K⁺-ATPase at 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 ammo-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([Δn/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 [Δn/c]CC was not affected by ouabain, although [Δn/c]CC can still bind [³H]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 α₁ 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 α₁ subunit.