Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1
Claire A. Ruddiman, Richard Peckham, Melissa A. Luse, Yen-Lin Chen, Maniselvan Kuppusamy, Bruce A. Corliss, P. Jordan Hall, Chien-Jung Lin, Shayn M. Peirce, Swapnil K. Sonkusare, Robert P. Mecham, Jessica E. Wagenseil, Brant E. Isakson
Claire A. Ruddiman, Richard Peckham, Melissa A. Luse, Yen-Lin Chen, Maniselvan Kuppusamy, Bruce A. Corliss, P. Jordan Hall, Chien-Jung Lin, Shayn M. Peirce, Swapnil K. Sonkusare, Robert P. Mecham, Jessica E. Wagenseil, Brant E. Isakson
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Research Article Vascular biology

Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1

Abstract

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 — an inwardly rectifying potassium channel that regulates membrane hyperpolarization — contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function.

Authors

Claire A. Ruddiman, Richard Peckham, Melissa A. Luse, Yen-Lin Chen, Maniselvan Kuppusamy, Bruce A. Corliss, P. Jordan Hall, Chien-Jung Lin, Shayn M. Peirce, Swapnil K. Sonkusare, Robert P. Mecham, Jessica E. Wagenseil, Brant E. Isakson

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Figure 5

PS inhibits PIP2 activation of Kir2.1.

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PS inhibits PIP2 activation of Kir2.1. (A) Average whole cell Kir2.1 cur...
(A) Average whole cell Kir2.1 currents at –140 mV in transfected HEK293T cells at baseline or with 100 μM Ba2+. Ba2+-sensitive currents were calculated by subtracting Ba2+ current from basal current. n = 5–7 cells per group. An ordinary 1-way ANOVA was performed with P < 0.05 significant effect for Ba2+-sensitive currents. Tukey’s multiple-comparison test was performed, where *P < 0.05. (B) Representative traces of Ba2+-sensitive currents, where basal current (black), 10 μM PIP2 (gray), 10 μM PS (magenta), and 10 μM PIP2 + 10μM PS (cyan). (C) Experimental timeline to assess PIP2 dilation in intact arteries. (D) Maximum change in diameter for each treatment group. n = 4–5 mice and n = 4–7 arteries per group. Changes in diameter for PS were taken from the same experiments as 10 μM PIP2 + 10 μM PS groups, where the maximum change in diameter was recorded within the 30-minute incubation prior to PIP2 treatment. An ordinary 1-way ANOVA was performed between PIP2, PS only, and PIP2 + PS with a significant interaction of P < 0.0005. Tukey’s multiple-comparison test was performed post hoc, where *P < 0.050 and ***P < 0.001. Other treatment groups were performed to ensure no unintended effects occurred with vehicles and to verify efficacy of Kir2.1 inhibitors; thus, they were excluded from the ANOVA. (E) Representative traces of inner diameter from pressure myography experiments treated with 10 μM PIP2, 10 μM PS, or 10 μM PIP2 + 10 μM. (F) Time point where maximum change in diameter was measured. (G) Dilation to 1 μM NS309 following a 5-minute washout period to assess EC function. (H) Constriction to 30 mM KCl to assess SMC function. One-way ANOVA was performed for F–H.