NA+-SENSITIVE, OUABAIN-SENSITIVE, CA2+-SENSITIVE, AND THAPSIGARGIN-SENSITIVE ATPASE ACTIVITY EXPRESSED IN CHIMERAS BETWEEN THE CALCIUM AND THE SODIUM-PUMP ALPHA-SUBUNITS
NA+-SENSITIVE, OUABAIN-SENSITIVE, CA2+-SENSITIVE, AND THAPSIGARGIN-SENSITIVE ATPASE ACTIVITY EXPRESSED IN CHIMERAS BETWEEN THE CALCIUM AND THE SODIUM-PUMP ALPHA-SUBUNITS
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DOI：
10.1073/pnas.91.13.6103
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发表时间：
1994-06-21
影响因子：
11.1
通讯作者：
TAKEYASU, K
中科院分区：
文献类型：
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作者：
ISHII, T;LEMAS, MV;TAKEYASU, K
以鸡肌质/内质网Ca ~（2+）-ATP酶（SERCA）为亲本分子，用鸡Na ~+，K ~+-ATP酶α（1）亚基的相应部分替换不同部分，鉴定了对这些P型ATP酶活性至关重要的Ca ~（2+）/毒胡萝卜素敏感结构域和Na ~+/哇巴因敏感结构域。在嵌合体[n/c]CC中，SERCA（同种型1）（SERCA 1）ATP酶的氨基末端氨基酸Met-1至Asp-162被Na+，K+-ATP酶α（1）的相应部分（Met-1-Asp-200）置换。在嵌合体CC[c/n]中，羧基末端氨基酸SERCA 1 ATP酶的Ser-830至COOH被相应的片段取代在嵌合体CNC中，Na+，K+-ATP酶α（1）亚基的中间部分（Leu-861至COOH）Na+，K+-ATP酶α（1）亚基（Gly-378-Lys-724）与SERCA 1 ATP酶的Gly-354-Lys-712交换。没有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，SERCA 1 ATP酶的Ile-163-Gly-354和Lys-712-Ser-830区段足以对Ca 2+和毒胡萝卜素敏感。在含Ca ~（2+）的测定培养基中加入Na ~+可进一步刺激[n/c]CC的SERCA_1-ATP酶活性，而CCC、CNC和[c/n] CC则无此活性。当[n/c]CC的氨基末端区域（Met-1-Leu-69）被删除（[Delta n/c]CC）时，Na+对SERCA 1-ATP酶活性的这种额外刺激被消除。在无Na+存在时，哇巴因抑制[n/c]CC的SERCA 1-ATP酶活性，而在Na+存在时，哇巴因刺激[n/c]CC的SERCA 1-ATP酶活性。另一方面，[Delta n/c]CC的ATP酶活性不受哇巴因的影响，尽管[Delta n/c]CC仍然可以结合[H-3]哇巴因。这些结果表明，位于Na+，K+-ATP酶α（1）亚基的限制性氨基末端区域（Met-1-Leu-69）内的一个独特的Na+敏感结构域（Na+传感器）调节ATP酶活性。Na+传感器还与α（1）亚基的Ala-70和Asp-200之间的主要哇巴因结合区域一起控制哇巴因的作用。
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.