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Journal of Lipid Research, Vol. 46, 504-515, March 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

pH-dependent domain formation in phosphatidylinositol polyphosphate/phosphatidylcholine mixed vesicles

Duane A. Redfern and Arne Gericke¹

Chemistry Department, Kent State University, Kent, OH 44242

¹ To whom the correspondence should be addressed. e-mail agericke{at}kent.edu

Phosphatidylinositol polyphosphates (PI-PPs) have been shown to mediate a large variety of physiological processes by attracting proteins to specific cellular sites. Such site-specific signaling requires local accumulation of PI-PPs, and in light of the rich headgroup functionality, it is conceivable that hydrogen bond formation between adjacent headgroups is a contributing factor to the formation of PI-PP-enriched domains. To explore the significance of hydrogen bond formation for the mutual interaction of PI-PPs, this study aims to characterize the pH-dependent phase behavior of phosphatidylcholine/phosphatidylinositol bisphosphate and trisphosphate mixed vesicles by differential scanning calorimetry, infrared transmission spectroscopy, and fluorescence resonance energy transfer measurements. For pH values >7–7.5, the experiments yielded results consistent with dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylinositol polyphosphate gel phase demixing, whereas for moderately acidic conditions, an enhanced mixing was observed. Similarly, this pH-dependent formation of PI-PP-enriched domains was also found for the physiologically important fluid phase.

The stability of PI-PP-enriched domains and to some extent the pH dependence of the domain formation was governed by the number as well as the position of the phosphomonoester groups at the inositol ring.

Abbreviations: Bodipy-PI-3,4-P₂, D(+)-sn-1-O-[1-[6'-[6-[((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl) amino]hexanoyl]amino]hexanoyl]2-hexanoylglyceryl D-myo-phosphatidylinositol-3,4-bisphosphate; Bodipy TR, [1-[6'-[6-[((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino] hexanoyl]amino]hexanoyl]; Bodipy TMR, [1-[6'-[6-[(4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionoyl)amino]hexanoyl]amino]hexanoyl]; CHES, 2-[N-cyclohexylamino] ethanesulfonic acid; DPPC, dipalmitoylphosphatidylcholine; DPPI, dipalmitoylphosphatidylinositol; DPPI-3,4-P₂, dipalmitoylphosphatidylinositol-3,4-bisphosphate; DPPI-3,5-P₂, dipalmitoylphosphatidylinositol-3,5-bisphosphate; DPPI-3,4,5-P₃, dipalmitoylphosphatidylinositol-3,4,5-trisphosphate; DPPI-4,5-P₂, dipalmitoylphosphatidylinositol-4,5-bisphosphate; DSC, differential scanning calorimetry; FRET, fluorescence resonance energy transfer; FTIR, Fourier transform infrared; MARCKS, myristoylated alanine-rich C kinase substrate; NBD-PC, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]sn-glycero-3-phosphocholine; PA, phosphatidic acid; PC, phosphatidylcholine; PI-3,4-P₂, phosphatidylinositol-3,4-bisphosphate; PI-3,4,5-P₃, phosphatidylinositol-3,4,5-trisphosphate; PI-3,5-P₂, phosphatidylinositol-3,5-bisphosphate; PI-4,5-P₂, phosphatidylinositol-4,5-bisphosphate; PI-PP, phosphatidylinositol polyphosphate; PI-xP, phosphatidylinositol monophosphate; POPC, palmitoyloleoylphosphatidylcholine; SAPI-3,4,5-P₃, stearoylarachidonoylphosphatidylinositol-3,4,5-trisphosphate; T_m, melting temperature; _a(CD₂), antisymmetric CD₂ stretching vibration band; _s(CH₂), symmetric CH₂ stretching band

Supplementary key words phosphoinositide • lipid demixing • calorimetry • infrared • fluorescence

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