Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells

Kenji Sugimoto, Motohiro Nishida, Masami Otsuka, Keisuke Makino, Katsutoshi Ohkubo, Yasuo Mori, Takashi Morii

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP3) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) δ. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP3 binding pocket for enhanced IP3 selectivity and for rapid and direct visualization of intracellular IP3 ≥ 0.5 μM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP3 production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP3 probe was extremely effective to quantitatively assess real-time physiological IP3 production via those pathways formed only in the intact cellular configuration.

Original languageEnglish
Pages (from-to)475-485
Number of pages11
JournalChemistry and Biology
Volume11
Issue number4
DOIs
Publication statusPublished - Apr 1 2004
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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