TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells

Xiaoxia Wang, Jennifer L. Pluznick, Peilin Wei, Babu J Padanilam, Steven Claude Sansom

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

Studies were performed to identify the molecular component responsible for store-operated Ca2+ entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise Ca2+-selective and -nonselective cation channels in a variety of cells, we screened TRPC1-TRPC7 with the use of molecular methods and the fura 2 method to determine their participation as components of the mesangial store-operated Ca2+ (SOC) channel. Using TRPC-specific primers and RT-PCR, we found that cultured MMC contained mRNA for TRPC1 and TRPC4 but not for TRPC2, TRPC3, TRPC5, TRPC6, and TRPC7. Immunocytochemical staining of MMC revealed predominantly cytoplasmic expression of TRPC1 and plasmalemmal expression of TRPC4. The role of TRPC4 in SOC was determined with TRPC4 antisense and fura 2 ratiometric measurements of intracellular Ca2+ concentration ([Ca2+]i). SOC was measured as the increase in [Ca2+]i after extracellular Ca2+ was increased from < 10 nM to 1 mM in the continued presence of thapsigargin. We found that TRPC4 antisense, which reduced plasmalemmal expression of TRPC4, inhibited SOC by 83%. Incubation with scrambled TRPC4 oligonucleotides did not affect SOC. Immunohistochemical staining identified expressed TRPC4 in the glomeruli of mouse renal sections. The results of RT-PCR performed to distinguish between TRPC4-α and TRPC4-β were consistent with expression of both isoforms in brain but with only TRPC4-α expression in MMC. These studies show that TRPC4-α may form the homotetrameric SOC in mouse mesangial cells.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume287
Issue number2 56-2
DOIs
StatePublished - Aug 1 2004

Fingerprint

Mesangial Cells
Fura-2
Staining and Labeling
Polymerase Chain Reaction
Thapsigargin
Oligonucleotides
Cations
Brain
Protein Isoforms
Kidney
Messenger RNA
Proteins

Keywords

  • Canonical transient receptor potential
  • Fura 2
  • Glomerulus
  • TRPC1
  • TRPC4-αTRPC-β4

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Cite this

TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells. / Wang, Xiaoxia; Pluznick, Jennifer L.; Wei, Peilin; Padanilam, Babu J; Sansom, Steven Claude.

In: American Journal of Physiology - Cell Physiology, Vol. 287, No. 2 56-2, 01.08.2004.

Research output: Contribution to journalArticle

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abstract = "Studies were performed to identify the molecular component responsible for store-operated Ca2+ entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise Ca2+-selective and -nonselective cation channels in a variety of cells, we screened TRPC1-TRPC7 with the use of molecular methods and the fura 2 method to determine their participation as components of the mesangial store-operated Ca2+ (SOC) channel. Using TRPC-specific primers and RT-PCR, we found that cultured MMC contained mRNA for TRPC1 and TRPC4 but not for TRPC2, TRPC3, TRPC5, TRPC6, and TRPC7. Immunocytochemical staining of MMC revealed predominantly cytoplasmic expression of TRPC1 and plasmalemmal expression of TRPC4. The role of TRPC4 in SOC was determined with TRPC4 antisense and fura 2 ratiometric measurements of intracellular Ca2+ concentration ([Ca2+]i). SOC was measured as the increase in [Ca2+]i after extracellular Ca2+ was increased from < 10 nM to 1 mM in the continued presence of thapsigargin. We found that TRPC4 antisense, which reduced plasmalemmal expression of TRPC4, inhibited SOC by 83{\%}. Incubation with scrambled TRPC4 oligonucleotides did not affect SOC. Immunohistochemical staining identified expressed TRPC4 in the glomeruli of mouse renal sections. The results of RT-PCR performed to distinguish between TRPC4-α and TRPC4-β were consistent with expression of both isoforms in brain but with only TRPC4-α expression in MMC. These studies show that TRPC4-α may form the homotetrameric SOC in mouse mesangial cells.",
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