Ishii T, Lemas M V, Takeyasu K
Department of Medical Biochemistry and Biotechnology Center, Ohio State University, Columbus 43210.
Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6103-7. doi: 10.1073/pnas.91.13.6103.
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 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 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 Ca(2+)-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 [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 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.
以鸡肌浆网/内质网Ca2+（SERCA）-ATP酶作为亲本分子，并将鸡Na+，K(+)-ATP酶α1亚基的相应部分替换其各个部分，确定了对这些P型ATP酶活性至关重要的Ca2+/毒胡萝卜素敏感域和Na+/哇巴因敏感域。在嵌合体[n/c]CC中，SERCA（同工型1）（SERCA1）ATP酶的氨基末端氨基酸Met-1至Asp-162被Na+，K(+)-ATP酶α1亚基的相应部分（Met-1-Asp-200）取代。在嵌合体CC[c/n]中，SERCA1 ATP酶羧基末端氨基酸（Ser-830至COOH）被Na+，K(+)-ATP酶α1亚基的相应片段（Leu-861至COOH）取代，在嵌合体CNC中，SERCA1 ATP酶的中间部分（Gly-354-Lys-712）与Na+，K(+)-ATP酶α1亚基（Gly-378-Lys-724）进行了交换。没有一个嵌合分子表现出任何可检测到的哇巴因敏感的Na+，K(+)-ATP酶活性，但它们确实表现出毒胡萝卜素敏感的Ca(2+)-ATP酶活性。因此，SERCA1 ATP酶的Ile-163-Gly-354和Lys-712-Ser-830片段足以实现Ca2+和毒胡萝卜素敏感性。在含有Ca2+的测定培养基中添加Na+可进一步刺激[n/c]CC的SERCA1-ATP酶活性，但不能刺激CCC、CNC或CC[c/n]的活性。当删除[n/c]CC的氨基末端区域（Met-1-Leu-69）（[δn/c]CC）时，Na+对SERCA1-ATP酶活性的这种额外刺激作用消失。在没有Na+的情况下，[n/c]CC的SERCA1-ATP酶活性受到哇巴因的抑制，而在有Na+的情况下，其活性受到该药物的刺激。另一方面，[δn/c]CC的ATP酶活性不受哇巴因影响，尽管[δn/c]CC仍能结合[3H]哇巴因。这些结果表明，位于Na+，K(+)-ATP酶α1亚基有限的氨基末端区域（Met-1-Leu-69）内的一个独特的Na(+)-敏感域（Na+传感器）调节ATP酶活性。Na+传感器还与α1亚基Ala-70和Asp-200之间的主要哇巴因结合区域协同控制哇巴因的作用。
