肌浆网/内质网Ca(2+) -ATP酶和Na+/K(+) -ATP酶的羧基末端区域控制着它们对K+的敏感性。

Carboxy-terminal regions of the sarcoplasmic/endoplasmic reticulum Ca(2+)- and the Na+/K(+)-ATPases control their K+ sensitivity.

作者信息

Ishii T, Hata F, Lemas M V, Fambrough D M, Takeyasu K

机构信息

Department of Medical Biochemistry, Ohio State University, Columbus 43210, USA.

出版信息

Biochemistry. 1997 Jan 14;36(2):442-51. doi: 10.1021/bi960644c.

DOI:10.1021/bi960644c

PMID:9003197

Abstract

The Na+,K(+)-ATPase and the sarcoplasmic/endoplasmic reticulum Ca(2+)-(SERCA-) ATPase belong to a family of P-type ATPases that undergo a cycle of conformational changes between the phosphorylated and dephosphorylated stages in an ion-specific manner. The ouabain-inhibitable Na+,K(+)-ATPase activity requires Na+ and K+. On the other hand, the Ca(2+)-dependent and thapsigargin-inhibitable activity of the SERCA-ATPase does not depend upon Na+ and K+ for its basal activity. However, the SERCA-ATPase and Ca(2+)-transport activities can be further activated either by K+ in a two-step fashion with high (ED50 approximately 20 mM) and low affinity (ED50 approximately 70 mM) or by Na+ in a one-step fashion with an ED50 value of approximately 50 mM. A chimera, in which the carboxy-terminal region (Leu861-COOH) of the Na+,K(+)-ATPase alpha 1 subunit replaced the corresponding region (Ser830-COOH) of the SERCA1-ATPase, lacked the low-affinity K+ activation of the SERCA-ATPase but displayed a higher-affinity (ED50 < 10 mM) activation by K+, similar to that of the Na+,K(+)-ATPase, whereas activation by Na+ was not affected. The replacement of the large cytosolic loop (Gly354-Lys712) and the amino-terminal regions (Met1-Asp162) of the SERCA1-ATPase with the corresponding portions of the Na+,K(+)-ATPase alpha 1 subunit did not affect the sensitivity of the SERCA-ATPase activity to K+. Thus, the carboxy-terminal regions of both the SERCA1 and the Na+,K(+)-ATPase alpha 1 subunit are critical for K+ sensitivity. Analysis of additional (Ca2+/Na+,K+)-ATPase chimeras demonstrated that the carboxy-terminal 102 amino acids (Phe920-Tyr1021) of the Na+/K(+)-ATPase alpha 1 subunit are sufficient to shift the K+ affinity for activation of the SERCA-ATPase without the beta subunit. No change in the two-step activation of SERCA-ATPase by K+ was seen when residues Thr871-Thr898 of the SERCA1-ATPase were replaced with residues Asn894-Ala919 of the Na+,K(+)-ATPase alpha 1 subunit, a region known to bind the Na+,K(+)-ATPase beta subunit [Lemas, M. V., et al. (1994) J. Biol. Chem. 269, 8255-8259]. Thus, the Na+,K(+)-ATPase subunit-assembly domain and the K(+)-sensitive region are distinct within the carboxy-terminal 161 amino acids of the Na+,K(+)-ATPase.

摘要

钠钾ATP酶和肌浆网/内质网钙ATP酶（SERCA）属于P型ATP酶家族，它们以离子特异性方式在磷酸化和去磷酸化阶段之间经历构象变化循环。哇巴因抑制的钠钾ATP酶活性需要钠离子和钾离子。另一方面，SERCA - ATP酶的钙依赖性和毒胡萝卜素抑制活性的基础活性并不依赖于钠离子和钾离子。然而，SERCA - ATP酶的活性和钙转运活性可以通过钾离子以两步方式进一步激活，高亲和力（半数有效浓度约为20 mM）和低亲和力（半数有效浓度约为70 mM），或者通过钠离子以一步方式激活，半数有效浓度值约为50 mM。一种嵌合体，其中钠钾ATP酶α1亚基的羧基末端区域（Leu861 - COOH）取代了SERCA1 - ATP酶的相应区域（Ser830 - COOH），缺乏SERCA - ATP酶的低亲和力钾离子激活，但表现出与钠钾ATP酶相似的更高亲和力（半数有效浓度<10 mM）的钾离子激活，而钠离子激活不受影响。用钠钾ATP酶α1亚基的相应部分替换SERCA1 - ATP酶的大胞质环（Gly354 - Lys712）和氨基末端区域（Met1 - Asp162），并不影响SERCA - ATP酶活性对钾离子的敏感性。因此，SERCA1和钠钾ATP酶α1亚基的羧基末端区域对于钾离子敏感性至关重要。对其他（钙/钠，钾）ATP酶嵌合体的分析表明，钠钾ATP酶α1亚基的羧基末端102个氨基酸（Phe920 - Tyr1021）足以在没有β亚基的情况下改变钾离子对SERCA - ATP酶激活的亲和力。当SERCA1 - ATP酶的Thr871 - Thr898残基被钠钾ATP酶α1亚基的Asn894 - Ala919残基取代时，未观察到SERCA - ATP酶被钾离子两步激活的变化，该区域已知可结合钠钾ATP酶β亚基[Lemas, M. V.,等人（1994年）《生物化学杂志》269, 8255 - 8259]。因此，钠钾ATP酶亚基组装结构域和钾离子敏感区域在钠钾ATP酶的羧基末端161个氨基酸内是不同的。