Plasma membrane Ca2+-ATPase and NCX1 Na+/Ca2+ exchanger expression in distal convoluted tubule cells

Clara E. Magyar, Kenneth E. White, Raul Rojas, Gerard Apodaca, Peter A. Friedman

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

The plasma membrane Ca2+-ATPase (PMCA) and the NCX1 Na+/Ca2+ exchanger regulate intracellular Ca2+ concentrations and mediate Ca2+ efflux in absorptive epithelial cells. We characterized the PMCA isoforms and subtypes expressed in mouse distal convoluted tubule (mDCT) cells and Na+/Ca2+ exchanger protein expression in mDCT cells. In lysates of mDCT cells, immunoprecipitation and Western blot analysis, performed with a monoclonal antibody to PMCA, revealed a 140-kDa protein consistent with PMCA. Laser-scanning confocal fluorescence microscopy indicated that PMCA and NCX1 expression is restricted to basolateral membranes only in confluent mDCT cells, because subconfluent cultures predominately express intracellular localizations. PMCA isoform-specific PCR primers generated appropriately sized products only for PMCA1 and PMCA4 from DCT cells but PMCA1-4 from whole mouse kidney. Assessment of splice site C within the calmodulin-binding domain demonstrated the presence of PMCA1b and PMCA4b mRNAs in mDCT cells. Northern blot analysis of mDCT cell RNA revealed transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4. We conclude that DCT cells express PMCA transcripts encoding PMCA1b and PMCA4b. Basolateral localization of the Na+/Ca2+ exchanger and PMCAs support the idea that multiple PMCA isoforms, in concert with the Na+/Ca2+ exchanger, mediate basal or hormone-stimulated Ca2+ efflux by distal tubules.

Original languageEnglish (US)
Pages (from-to)F29-F40
JournalAmerican Journal of Physiology - Renal Physiology
Volume283
Issue number1 52-1
StatePublished - Jul 4 2002

Fingerprint

Calcium-Transporting ATPases
Cell Membrane
Protein Isoforms
Calmodulin
Fluorescence Microscopy
Immunoprecipitation
Confocal Microscopy
Northern Blotting
Proteins
Lasers
Western Blotting
Epithelial Cells
Monoclonal Antibodies
Hormones
RNA
Kidney
Polymerase Chain Reaction
Messenger RNA
Membranes

Keywords

  • Calcium transport
  • Confocal fluorescence microscopy
  • Kidney
  • Plasma membrane calcium-adenosine 5′-triphosphatase
  • Sodium-calcium exchange

ASJC Scopus subject areas

  • Physiology

Cite this

Plasma membrane Ca2+-ATPase and NCX1 Na+/Ca2+ exchanger expression in distal convoluted tubule cells. / Magyar, Clara E.; White, Kenneth E.; Rojas, Raul; Apodaca, Gerard; Friedman, Peter A.

In: American Journal of Physiology - Renal Physiology, Vol. 283, No. 1 52-1, 04.07.2002, p. F29-F40.

Research output: Contribution to journalArticle

Magyar, Clara E. ; White, Kenneth E. ; Rojas, Raul ; Apodaca, Gerard ; Friedman, Peter A. / Plasma membrane Ca2+-ATPase and NCX1 Na+/Ca2+ exchanger expression in distal convoluted tubule cells. In: American Journal of Physiology - Renal Physiology. 2002 ; Vol. 283, No. 1 52-1. pp. F29-F40.
@article{096ce4103b3a4d0988279ff92a1fc5d3,
title = "Plasma membrane Ca2+-ATPase and NCX1 Na+/Ca2+ exchanger expression in distal convoluted tubule cells",
abstract = "The plasma membrane Ca2+-ATPase (PMCA) and the NCX1 Na+/Ca2+ exchanger regulate intracellular Ca2+ concentrations and mediate Ca2+ efflux in absorptive epithelial cells. We characterized the PMCA isoforms and subtypes expressed in mouse distal convoluted tubule (mDCT) cells and Na+/Ca2+ exchanger protein expression in mDCT cells. In lysates of mDCT cells, immunoprecipitation and Western blot analysis, performed with a monoclonal antibody to PMCA, revealed a 140-kDa protein consistent with PMCA. Laser-scanning confocal fluorescence microscopy indicated that PMCA and NCX1 expression is restricted to basolateral membranes only in confluent mDCT cells, because subconfluent cultures predominately express intracellular localizations. PMCA isoform-specific PCR primers generated appropriately sized products only for PMCA1 and PMCA4 from DCT cells but PMCA1-4 from whole mouse kidney. Assessment of splice site C within the calmodulin-binding domain demonstrated the presence of PMCA1b and PMCA4b mRNAs in mDCT cells. Northern blot analysis of mDCT cell RNA revealed transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4. We conclude that DCT cells express PMCA transcripts encoding PMCA1b and PMCA4b. Basolateral localization of the Na+/Ca2+ exchanger and PMCAs support the idea that multiple PMCA isoforms, in concert with the Na+/Ca2+ exchanger, mediate basal or hormone-stimulated Ca2+ efflux by distal tubules.",
keywords = "Calcium transport, Confocal fluorescence microscopy, Kidney, Plasma membrane calcium-adenosine 5′-triphosphatase, Sodium-calcium exchange",
author = "Magyar, {Clara E.} and White, {Kenneth E.} and Raul Rojas and Gerard Apodaca and Friedman, {Peter A.}",
year = "2002",
month = "7",
day = "4",
language = "English (US)",
volume = "283",
pages = "F29--F40",
journal = "American Journal of Physiology - Renal Physiology",
issn = "0363-6127",
number = "1 52-1",

}

TY - JOUR

T1 - Plasma membrane Ca2+-ATPase and NCX1 Na+/Ca2+ exchanger expression in distal convoluted tubule cells

AU - Magyar, Clara E.

AU - White, Kenneth E.

AU - Rojas, Raul

AU - Apodaca, Gerard

AU - Friedman, Peter A.

PY - 2002/7/4

Y1 - 2002/7/4

N2 - The plasma membrane Ca2+-ATPase (PMCA) and the NCX1 Na+/Ca2+ exchanger regulate intracellular Ca2+ concentrations and mediate Ca2+ efflux in absorptive epithelial cells. We characterized the PMCA isoforms and subtypes expressed in mouse distal convoluted tubule (mDCT) cells and Na+/Ca2+ exchanger protein expression in mDCT cells. In lysates of mDCT cells, immunoprecipitation and Western blot analysis, performed with a monoclonal antibody to PMCA, revealed a 140-kDa protein consistent with PMCA. Laser-scanning confocal fluorescence microscopy indicated that PMCA and NCX1 expression is restricted to basolateral membranes only in confluent mDCT cells, because subconfluent cultures predominately express intracellular localizations. PMCA isoform-specific PCR primers generated appropriately sized products only for PMCA1 and PMCA4 from DCT cells but PMCA1-4 from whole mouse kidney. Assessment of splice site C within the calmodulin-binding domain demonstrated the presence of PMCA1b and PMCA4b mRNAs in mDCT cells. Northern blot analysis of mDCT cell RNA revealed transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4. We conclude that DCT cells express PMCA transcripts encoding PMCA1b and PMCA4b. Basolateral localization of the Na+/Ca2+ exchanger and PMCAs support the idea that multiple PMCA isoforms, in concert with the Na+/Ca2+ exchanger, mediate basal or hormone-stimulated Ca2+ efflux by distal tubules.

AB - The plasma membrane Ca2+-ATPase (PMCA) and the NCX1 Na+/Ca2+ exchanger regulate intracellular Ca2+ concentrations and mediate Ca2+ efflux in absorptive epithelial cells. We characterized the PMCA isoforms and subtypes expressed in mouse distal convoluted tubule (mDCT) cells and Na+/Ca2+ exchanger protein expression in mDCT cells. In lysates of mDCT cells, immunoprecipitation and Western blot analysis, performed with a monoclonal antibody to PMCA, revealed a 140-kDa protein consistent with PMCA. Laser-scanning confocal fluorescence microscopy indicated that PMCA and NCX1 expression is restricted to basolateral membranes only in confluent mDCT cells, because subconfluent cultures predominately express intracellular localizations. PMCA isoform-specific PCR primers generated appropriately sized products only for PMCA1 and PMCA4 from DCT cells but PMCA1-4 from whole mouse kidney. Assessment of splice site C within the calmodulin-binding domain demonstrated the presence of PMCA1b and PMCA4b mRNAs in mDCT cells. Northern blot analysis of mDCT cell RNA revealed transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4. We conclude that DCT cells express PMCA transcripts encoding PMCA1b and PMCA4b. Basolateral localization of the Na+/Ca2+ exchanger and PMCAs support the idea that multiple PMCA isoforms, in concert with the Na+/Ca2+ exchanger, mediate basal or hormone-stimulated Ca2+ efflux by distal tubules.

KW - Calcium transport

KW - Confocal fluorescence microscopy

KW - Kidney

KW - Plasma membrane calcium-adenosine 5′-triphosphatase

KW - Sodium-calcium exchange

UR - http://www.scopus.com/inward/record.url?scp=0036088309&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036088309&partnerID=8YFLogxK

M3 - Article

C2 - 12060584

AN - SCOPUS:0036088309

VL - 283

SP - F29-F40

JO - American Journal of Physiology - Renal Physiology

JF - American Journal of Physiology - Renal Physiology

SN - 0363-6127

IS - 1 52-1

ER -