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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 (PIP 2) binding on Kir2. 1. We found that Kir2. 1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2. 1. Electrophysiology …