Biosensors to measure inositol 1,4,5-trisphosphate concentration in living cells with spatiotemporal resolution

Timothy P. Remus, Aleksey V. Zima, Julie B C Bossuyt, Dan J. Bare, Jody L. Martin, Lothar A. Blatter, Donald M Bers, Gregory A. Mignery

Research output: Contribution to journalArticle

77 Scopus citations

Abstract

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling.Weconstructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an ∼11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent Kd = 31.3 ± 6.7 nM InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to ∼30 nM). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3) in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.

Original languageEnglish (US)
Pages (from-to)608-616
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number1
DOIs
StatePublished - Jan 6 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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