TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells.
Studies were performed to identify the molecular component responsible for store-operated Ca(2+) entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise Ca(2+)-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 Ca(2+) (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 Ca(2+) concentration ([Ca(2+)](i)). SOC was measured as the increase in [Ca(2+)](i) after extracellular Ca(2+) 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-alpha and TRPC4-beta were consistent with expression of both isoforms in brain but with only TRPC4-alpha expression in MMC. These studies show that TRPC4-alpha may form the homotetrameric SOC in mouse mesangial cells.
Wang
Xiaoxia
X
Department of Physiology and Biophysics, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA.
Pluznick
Jennifer L
JL
Wei
Peilin
P
Padanilam
Babu J
BJ
Sansom
Steven C
SC
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D016373:DNA, Antisense; D005456:Fluorescent Dyes; D016257:Fura-2; D005920:Glomerular Mesangium / Q000166:cytology* / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050052:TRPC Cation Channels
D015220:Calcium Channels; D016373:DNA, Antisense; D005456:Fluorescent Dyes; D050052:TRPC Cation Channels; D002118:Calcium; D016257:Fura-2