Molecular dissection of Ca2+ efflux in immortalized proximal tubule cells

Kenneth White, Frank A. Gesek, Teresa Nesbitt, Marc K. Drezner, Peter A. Friedman

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger participate in regulating cell function by maintaining proper intracellular Ca2+ concentrations ([Ca2+](i)). In renal epithelial cells these proteins have been additionally implicated in cellular calcium absorption. The purpose of the present studies was to determine the Ca2+ extrusion mechanisms in cells derived from the proximal tubule. Homology-based RT-PCR was used to amplify PMCA transcripts from RNA isolated from mouse cell lines originating from the S1, S2, and S3 proximal tubule segments. S1, S2, and S3 cells exhibited only PMCA1 and PMCA4 products. PCR product identity was confirmed by sequence analysis. Northern analysis of proximal tubule cell RNAs revealed appropriate transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4, but were negative for PMCA2 and PMCA3. Western analysis with a monoclonal antibody to PMCA showed that all proximal cell lines expressed a reacting plasma membrane protein of 140 kD, the reported PMCA molecular mass. Na+/Ca2+ exchanger (NCX1) mRNA expression, analyzed by RT-PCR, protein expression by Western analysis, and functional exchange activity were uniformly absent from all proximal tubule cell lines. These observations support the idea that immortalized cells derived from the proximal tubule express PMCA1 and PMCA4, which may serve as the primary mechanism of cellular Ca2+ efflux.

Original languageEnglish (US)
Pages (from-to)217-228
Number of pages12
JournalJournal of General Physiology
Volume109
Issue number2
DOIs
StatePublished - Feb 1997
Externally publishedYes

Fingerprint

Calcium-Transporting ATPases
Dissection
Cell Membrane
Cell Line
Polymerase Chain Reaction
RNA
Sequence Analysis
Blood Proteins
Membrane Proteins
Proteins
Epithelial Cells
Monoclonal Antibodies
Calcium
Kidney
Messenger RNA

Keywords

  • Ca-ATPase
  • calcium transport
  • kidney
  • Na/Ca exchange
  • PMCA

ASJC Scopus subject areas

  • Physiology

Cite this

Molecular dissection of Ca2+ efflux in immortalized proximal tubule cells. / White, Kenneth; Gesek, Frank A.; Nesbitt, Teresa; Drezner, Marc K.; Friedman, Peter A.

In: Journal of General Physiology, Vol. 109, No. 2, 02.1997, p. 217-228.

Research output: Contribution to journalArticle

White, Kenneth ; Gesek, Frank A. ; Nesbitt, Teresa ; Drezner, Marc K. ; Friedman, Peter A. / Molecular dissection of Ca2+ efflux in immortalized proximal tubule cells. In: Journal of General Physiology. 1997 ; Vol. 109, No. 2. pp. 217-228.
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