Na+-, ouabain-, Ca2+-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump α subunits

Toshiaki Ishii, Mario Victor Lemas, Kunio Takeyasu

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)6103-6107
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number13
StatePublished - Jun 21 1994

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Sodium-Potassium-Exchanging ATPase
Thapsigargin
Ouabain
Adenosine Triphosphatases
Calcium
Calcium-Transporting ATPases
Sarcoplasmic Reticulum
Endoplasmic Reticulum
Chickens
Amino Acids
sodium-translocating ATPase
Protein Isoforms

Keywords

  • Ca-ATPase
  • Cardiac glycoside
  • Chimeric molecule
  • Na,K-ATPase
  • Thapsigargin

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

@article{aef3f6d84baf4667aed02f0d68e6b7e0,
title = "Na+-, ouabain-, Ca2+-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump α subunits",
abstract = "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.",
keywords = "Ca-ATPase, Cardiac glycoside, Chimeric molecule, Na,K-ATPase, Thapsigargin",
author = "Toshiaki Ishii and Lemas, {Mario Victor} and Kunio Takeyasu",
year = "1994",
month = "6",
day = "21",
language = "English (US)",
volume = "91",
pages = "6103--6107",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "13",

}

TY - JOUR

T1 - Na+-, ouabain-, Ca2+-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump α subunits

AU - Ishii, Toshiaki

AU - Lemas, Mario Victor

AU - Takeyasu, Kunio

PY - 1994/6/21

Y1 - 1994/6/21

N2 - 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.

AB - 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.

KW - Ca-ATPase

KW - Cardiac glycoside

KW - Chimeric molecule

KW - Na,K-ATPase

KW - Thapsigargin

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M3 - Article

VL - 91

SP - 6103

EP - 6107

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 13

ER -