Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface

Megan E. Cavet, Shafinaz Akhter, Fermin Sanchez De Medina, Mark Donowitz, Chung Ming Tse

Research output: Contribution to journalArticle

Abstract

NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na+/H+ exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity (V(max)) by 22Na+ uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and 99.2 ± 9.1 s-1, when V(max) was determined using 22Na uptake at 22°C and 742 ± 47,459 ± 16, and 609 ± 39 s-1 when V(max) was determined using fluorometry at 37°C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume277
Issue number6 46-6
StatePublished - 1999

Fingerprint

Sodium-Hydrogen Antiporter
Viruses
Glycoproteins
Vesicular Stomatitis
Maltose-Binding Proteins
Fibroblasts
Epitopes
Biotinylation
Genes
Fluorometry
Chemical analysis
Proteins

Keywords

  • Cell surface biotinylation
  • PS120 fibroblasts
  • Quantitative Western analysis
  • Sodium/hydrogen antiporter

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface. / Cavet, Megan E.; Akhter, Shafinaz; De Medina, Fermin Sanchez; Donowitz, Mark; Tse, Chung Ming.

In: American Journal of Physiology - Cell Physiology, Vol. 277, No. 6 46-6, 1999.

Research output: Contribution to journalArticle

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abstract = "NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na+/H+ exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity (V(max)) by 22Na+ uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and 99.2 ± 9.1 s-1, when V(max) was determined using 22Na uptake at 22°C and 742 ± 47,459 ± 16, and 609 ± 39 s-1 when V(max) was determined using fluorometry at 37°C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.",
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AU - Cavet, Megan E.

AU - Akhter, Shafinaz

AU - De Medina, Fermin Sanchez

AU - Donowitz, Mark

AU - Tse, Chung Ming

PY - 1999

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N2 - NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na+/H+ exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity (V(max)) by 22Na+ uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and 99.2 ± 9.1 s-1, when V(max) was determined using 22Na uptake at 22°C and 742 ± 47,459 ± 16, and 609 ± 39 s-1 when V(max) was determined using fluorometry at 37°C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.

AB - NHE1, NHE2, and NHE3 are well-characterized cloned members of the mammalian Na+/H+ exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbers of NHE1, NHE2, and NHE3 measured in the same cell system: PS120 fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitope tagged with vesicular stomatitis virus glycoprotein (VSVG). The following characteristics were determined on the same passage of cells transfected with NHE1V, NHE2V, or NHE3V: 1) maximal reaction velocity (V(max)) by 22Na+ uptake and fluorometery, 2) total amount of NHE protein by quantitative Western analysis with internal standards of VSVG-tagged maltose-binding protein, and 3) cell surface expression by cell surface biotinylation. Cell surface expression (percentage of total NHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despite these divergent cell surface expression levels, turnover numbers for NHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and 99.2 ± 9.1 s-1, when V(max) was determined using 22Na uptake at 22°C and 742 ± 47,459 ± 16, and 609 ± 39 s-1 when V(max) was determined using fluorometry at 37°C). These data indicate that, in the same cell system, intrinsic properties that determine turnover number are conserved among NHE1, NHE2, and NHE3.

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KW - Quantitative Western analysis

KW - Sodium/hydrogen antiporter

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