The role of endogenous human Trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells

Xiaoyan Wu, György Babnigg, Tatiana Zagranichnaya, Mitchel L. Villereal

Research output: Contribution to journalArticlepeer-review

82 Scopus citations


We utilized 2-aminoethyoxydiphenyl borane, an agent that blocks store-operated Ca 2+ entry, as well as an antisense approach to characterize endogenous Ca 2+ entry pathways in HEK-293 cells. The thapsigargin- and carbachol-induced, but not the 1-oleolyl-2-acytyl-sn-glycerol (OAG)-induced, entry was blocked by 2-aminoethyoxydiphenyl borane. Both reverse transcriptase-PCR and Western blot analyses demonstrated endogenous expression for HTRP1, HTRP3, and HTRP4 and specific suppression of mRNA levels and Trp protein levels in cells stably expressing antisense constructs. Expression of HTRP4 antisense inhibited 35% of the carbachol (CCh)-stimulated Ba 2+ entry and 46% of the OAG-stimulated Sr 2+ entry but in contrast had no effect on the thapsigargin-stimulated Ba 2+ or Sr 2+ entry. HTRP3 antisense reduced, while HTRP1 antisense had no effect on, OAG-induced Sr 2+ entry. Of greater importance, HTRP4 antisense expression, but not HTRP3 antisense expression, blocked the sustained Ca 2+ oscillations produced by low doses of CCh (15 μM), arguing that receptor-stimulated rather than store-operated channels are involved in these sustained oscillations. HTRP4 antisense also inhibited 75% of the arachidonic acid-induced Ca 2+ entry. In summary, these data suggest that HTRP4 proteins in HEK-293 cells, differing from HTRP3 and HTRP1 proteins, do not serve as functional subunits of store-operated channels but do function as subunits for CCh- and OAG-stimulated channels. Furthermore, evidence is provided for the first time for the involvement of a Trp isoform (HTRP4) in the formation of the channel responsible for both arachidonic acid-induced Ca 2+ entry and the Ca 2+ entry needed to sustain long term Ca 2+ oscillations induced by low doses of carbachol.

Original languageEnglish
Pages (from-to)13597-13608
Number of pages12
JournalJournal of Biological Chemistry
Issue number16
StatePublished - Apr 19 2002

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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